THREOSE NUCLEIC ACID ANTISENSE OLIGONUCLEOTIDES AND METHODS THEREOF

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International Patent Application No. PCT/EP2023/073466 filed Aug. 28, 2023, which claims the benefit of priority to European Patent Application No. 22192656.1 filed Aug. 29, 2022, each of which are incorporated herein by reference.

SEQUENCE LISTING

The present application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Nov. 13, 2024, is named “067211.023US1 SeqListing .xml” and is 44 kilobytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to antisense oligonucleotides comprising one or more α-L-threofuranosyl (TNA) nucleosides linked to an adjacent nucleoside by a phosphodiester internucleoside linkage, as well as methods to modulate the properties of antisense oligonucleotides by the introduction of such TNA nucleosides. The invention is particularly applicable for antisense gapmer oligonucleotides.

BACKGROUND OF THE INVENTION

Synthetic oligonucleotides as therapeutic agents have witnessed remarkable progress over recent years leading to a broad portfolio of clinically validated molecules acting by diverse mechanisms including antisense oligonucleotides such as ribonuclease H (RNase H) activating gapmers, splice switching oligonucleotides, micro-RNA inhibitors, small interfering RNA (siRNA) and aptamers (S. T. Crooke, Antisense drug technology: principles, strategies, and applications, 2nd ed. Boca Raton, FL: CRC Press, 2008).

Arguably one of the most successful modifications is the introduction of phosphorothioate (PS) linkages, where one of the non-bridging phosphate oxygen atoms is replaced with a sulfur atom (Eckstein, Antisense and Nucleic Acid Drug Development 2009; 10:117-121). Phosphorothioate oligodeoxynucleotides show an increased protein binding as well as a distinctly higher stability to nucleolytic degradation and thus a higher half-life in plasma, tissues and cells than their unmodified phosphodiester analogues. For example, a recent review (Crooke et al., Nucleic Acids Research 2020; 48 (10): 5235-5253) described the PS moiety as being the primary determinant of the distribution of single stranded antisense oligonucleotides after all routes of administration. Other modifications include Locked Nucleic Acids (LNAs) as well as a variety of other modified nucleosides. TNAs, for example, have been used, e.g., in double-stranded siRNA molecules and in the form of oligomers (Matsuda et al., XXIII International Round Table on Nucleosides, Nucleotides and Nucleic acids; 2018, Liu et al., ACS Appl. Mater. Interfaces 2018; 10:9736-9743, WO 2012/078536, WO 2012/118911, and WO 2013/179292).

There remains, however, a need for stable, safe, and efficient antisense oligonucleotide-based therapeutic agents.

SUMMARY OF THE INVENTION

It has been found by the present inventor(s) that one or more α-L-threofuranosyl (TNA) nucleosides can be introduced into antisense oligonucleotides via linkages other than PS linkages, particularly into antisense gapmer oligonucleotides, to modulate properties of the antisense oligonucleotides. Surprisingly, TNA nucleosides, particularly when introduced into gapmer designs via phosphodiester (PO) internucleoside linkages as described herein, can yield potent molecules with favourable properties for therapeutic use.

Accordingly, the present invention relates to antisense oligonucleotides comprising at least one TNA nucleoside linked to at least one adjacent nucleoside via an internucleoside linkage different from a PS linkage, particularly to antisense gapmer oligonucleotides comprising at least one such TNA nucleoside. Particularly preferred are PO internucleoside linkages. A TNA nucleoside linked to at least one adjacent nucleoside via a PO linkage can hereinafter be referred to as a TNA (PO) nucleoside. Contemplated TNA (PO) nucleosides include those linked by a 2′-PO linkage, a 3′-PO linkage, or both; hereinafter referred to as 2′-PO linked, 3′-PO linked, and 2′,3′-PO linked TNA nucleosides, respectively.

The invention also relates to an antisense gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I) which is capable of recruiting RNase H, wherein the contiguous nucleotide sequence comprises at least one TNA nucleoside which is linked to an adjacent nucleoside by a linkage different than a PS internucleoside linkage, such as by a PO internucleoside linkage.

The invention also relates to an antisense gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I) which is capable of recruiting ribonuclease (RNase) H, wherein

• • G is a gap region of up to 18 linked nucleosides which comprises at least 3 contiguous DNA nucleosides,
  • each of F and F′ is a flanking region of up to 15 linked nucleosides which independently comprises or consists of 1 to 15 sugar-modified nucleosides,
  • at least one of F, F′ and G comprises a sugar-modified nucleoside which is an α-L-threofuranosyl (TNA) nucleoside and which is linked to an adjacent nucleoside by an internucleoside linkage different than a PS internucleoside linkage, such as by a PO internucleoside linkage.

The invention also relates to a conjugate comprising an antisense gapmer oligonucleotide according to the invention and at least one conjugate moiety covalently attached to the antisense gapmer oligonucleotide, optionally via a linker.

The invention also relates to a pharmaceutically acceptable salt of an antisense gapmer oligonucleotide or conjugate according to the invention.

The invention also relates to a pharmaceutical composition comprising an antisense gapmer oligonucleotide, conjugate, or pharmaceutically acceptable salt according to the invention, and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

The invention also relates to an antisense oligonucleotide, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition according to the invention, for use as a medicament.

The invention also relates to a method of preparing a modified version of a parent antisense gapmer oligonucleotide, wherein the parent antisense gapmer comprises a contiguous nucleotide sequence of formula 5′ F-G-F′ 3′ (I) which is capable of recruiting RNase H, wherein G is a gap region of 5 to 18 linked DNA nucleosides and each of F and F′ is a flanking region of up to 8 linked nucleosides which independently comprises or consists of 1 to 8 sugar-modified nucleosides other than TNA nucleosides, and wherein, in the modified version, at least one nucleoside in F, F′, and/or G of the parent antisense gapmer oligonucleotide has been replaced with a TNA nucleoside linked to an adjacent nucleoside by an internucleoside linkage different than a PS internucleoside linkage, such as by a PO internucleoside linkage,

• • the method comprising the step of manufacturing the modified antisense gapmer oligonucleotide by reacting nucleotide units to form covalently linked contiguous nucleotide units comprised in the oligonucleotide, wherein at least one of the nucleotide units comprises a TNA nucleoside, and,
  • optionally purifying or isolating the modified antisense gapmer oligonucleotide.

The invention also relates to an antisense gapmer oligonucleotide obtained or obtainable by the method of the invention.

The invention also relates to the use of a TNA nucleotide in the preparation of an antisense gapmer oligonucleotide according to the invention.

Further details of these and other aspects and embodiments of the invention are provided in the following detailed disclosure and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Relative MALAT1 RNA expression in C57BL/6J mice. Antisense oligonucleotides were administered at a dose of 10 mg/kg by subcutaneous injection and RNA levels on day 9 determined. See Example 6 for details.

FIGS. 2A-H. Tissue concentrations and pharmacokinetic/pharmacodynamic (PK/PD) relationships. Concentrations of antisense oligonucleotides in liver (A), kidney (B), lung (C), and muscle (D) are shown, as well as PK/PD relationships for liver (E), kidney (F), lung (G) and muscle (H). See Example 6 for details.

DETAILED DISCLOSURE

Definitions

In order that the present invention may be more readily understood, certain terms are defined and described in the following.

Throughout this description, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components) but not the exclusion of any other integer (or components) or group of integers (or components).

Oligonucleotide

The term “oligonucleotide” as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides, including modified nucleosides or nucleotides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to an oligonucleotide sequence, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide of the invention is man-made, chemically synthesized, and is typically purified or isolated. The nucleosides may be linked by phosphodiester (PO) linkages or by modified internucleoside linkages.

Antisense Oligonucleotides

The term “antisense oligonucleotide” as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, particularly to a contiguous sequence on a target nucleic acid. The contemplated antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs or short hairpin RNAs (shRNAs). Preferably, the antisense oligonucleotides of the present invention are single stranded. It is understood that single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide) if the degree of intra or inter self-complementarity is more than 50% across of the full length of the oligonucleotide.

Contiguous Nucleotide Sequence

The term “contiguous nucleotide sequence” refers to the region of the oligonucleotide, which is complementary to a target nucleic acid. The term is used interchangeably herein with the term “contiguous nucleobase sequence” and the term “oligonucleotide motif sequence”. For example, all the nucleotides of the oligonucleotide may constitute the contiguous nucleotide sequence. Alternatively, the oligonucleotide may comprise the contiguous nucleotide sequence, such as an F-G-F′ gapmer region, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. Advantageously, the contiguous nucleotide sequence is 100% complementary to the target nucleic acid.

Nucleotides

Nucleotides are the building blocks of oligonucleotides and polynucleotides, and include, for the purposes of the present invention, both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as “units” or “monomers”.

Nucleobase

The term nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moiety present in nucleosides and nucleotides, which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases, which may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45. page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37, 1.4.1.

The nucleobase moiety can optionally be modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2′-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.

The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g., A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in some oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine (^mC).

Modified Nucleoside

The term “modified nucleoside” or “nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo) base moiety. Preferably, the modified nucleoside comprises a modified sugar moiety. The term “modified nucleoside” may also be used herein interchangeably with the term “nucleoside analogue” or modified “unit” or modified “monomer”. Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson-Crick base pairing.

Sugar-Modified Nucleosides

The antisense oligonucleotides of the invention may comprise one or more nucleosides, which have a modified sugar moiety, i.e., a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.

Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those where the ribose ring structure is modified, e.g., by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2′—OH group naturally found in DNA and RNA nucleosides. Substituents may, for example, be introduced at the 2′, 3′, 4′ or 5′ positions.

Non-limiting examples of modified sugar moieties include the following:

• • α-L-threofuranosyl (as in threose nucleic acid; TNA),
  • 2′-methoxy-ribose (2′-OMe),
  • 2′-O-methoxyethyl-ribose (2′-O-MOE),
  • 5′-methyl-2′-O-methoxyethyl ribose (5′-Me-2′-O-MOE),
  • 2′-O-[2-(methylthio)ethyl]-ribose (2′-O-MTE),
  • 2-(N-methylcarbamoyl)-ethyl]-ribose (2′-O-MCE),
  • 2′-O-[2-(methylamino)-2-oxoethyl]-ribose (2′-O-NMA),
  • 2′-deoxy-2′-fluoro-ribose (as in 2′-deoxy-2′-fluororibo-nucleic acid; 2′-F-RNA),
  • 2′-fluoro-2′-arabinose (as in 2′-fluoro-2′-arabinose nucleic acid; 2′-F-ANA),
  • 2′-O-benzyl-ribose,
  • oxy β-D-locked ribose (as in 3-D-LNA),
  • amino β-D-locked ribose (as in amino-β-D-LNA),
  • thio β-D-locked ribose (as in thio-β-D-LNA),
  • oxy β-L-locked ribose (as in β-L-LNA),
  • amino β-L-locked ribose (as in amino-β-L-LNA),
  • thio β-L-locked ribose (as in thio-β-L-LNA),
  • oxy α-L-locked ribose (as in α-L-LNA),
  • amino α-L-locked ribose (as in amino-α-L-LNA),
  • thio α-L-locked ribose (as in thio-α-L-LNA),
  • 2′, 4′-constrained 2′-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt), tricyclo-deoxyribose (as in tricyclo-deoxyribose DNA; TcDNA),
  • 3′-deoxy-ribose (as in 3′-deoxy-ribose DNA; 3′-DNA),
  • unlocked ribose (as in unlocked nucleic acid; UNA),
  • glycol (as in glycol nucleic acid; GNA),
  • hexitol (as in hexitol nucleic acid; HNA),
  • 3′-fluoro hexitol (as in 3′-fluoro hexitol nucleic acid; FHNA),
  • 3′-arabino-fluoro hexitol (as in 3′-arabino-fluoro hexitol nucleic acid; Ara-FHNA), cyclohexene (as in cyclohexene nucleic acid; CeNA),
  • fluoro-cyclohexenenyl (as in 2′-fluoro-cyclohexenyl nucleic acid; F-CeNA),
  • serinol (as in serinol nucleic acid; SNA),
  • 2′-O,4′-C-ethylene bridged ribose (as in 2′-O,4′-C-ethylene linked nucleic acid;
  • ENA), acyclic (L)-threoninol (as in acyclic (L)-threoninol nucleic acid; aTNA),
  • 2′,4′-constrained 2′-O-methoxyethyl ribose (as in cMOE), and
  • 7′,5′-alpha-bicyclo sugar unit (as in 7′,5′-alpha-bicyclo DNA; bcDNA).

Unless otherwise specified or contradicted by context, the term “MOE” may herein refer to any nucleoside which comprises an O-methoxyethyl-group at the 2′ position of the ribose ring, including, but not limited to, 2′-O-MOE and 5′-Me-2′-O-MOE.

Threose Nucleic Acids (TNA)

As used herein, an “α-L-threofuranosyl nucleoside”, “α-L-threose nucleic acid nucleoside”, “TNA nucleoside”, “TNA-modified nucleoside”, “TNA unit”, “TNA moiety” and the like refers to a sugar-modified nucleoside which comprises an α-L-threofuranosyl moiety.

TNA nucleosides are linked to adjacent nucleosides by (2′->3′) internucleoside linkages, e.g., phosphodiester (PO) or modified internucleoside linkages, as illustrated below for two adjacent TNA nucleosides.

A sugar-modified nucleoside which comprises an α-L-threofuranosyl moiety and is linked to at least one adjacent nucleoside via a PO linkage can be referred to herein as a “α-L-threofuranosyl (PO) nucleoside”, “α-L-threose nucleic acid (PO) nucleoside”, “TNA (PO) nucleoside”, “TNA (PO)-modified nucleoside”, “TNA (PO) unit”, “TNA (PO) moiety” and the like. Contemplated TNA (PO) nucleosides include those linked by a 2′-PO linkage, a 3′-PO linkage, or both; hereinafter referred to as a “2′-PO linked TNA nucleoside”, “3′-PO linked TNA nucleoside”, and “2′,3′-PO linked TNA nucleoside,” respectively.

When the nucleobase (B) is cytosine, the TNA or TNA (PO) nucleoside is advantageously a 5-methyl-cytosine (mC) TNA or TNA (PO) nucleoside.

2′-Sugar-Modified Nucleosides

A 2′-sugar modified nucleoside is a nucleoside which has a substituent other than H or —OH at the 2′-position (2′-substituted nucleoside). This includes a nucleoside which comprises a 2′-linked biradical capable of forming a bridge between the 2′-carbon and a second carbon in the ribose ring, such as LNA (2′-4′ bridged) nucleosides.

For the purpose of the present disclosure, a TNA or TNA (PO) nucleoside is not a 2′-substituted nucleoside.

Numerous 2′ substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, a 2′-modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide. Examples of 2′-substituted modified nucleosides are 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (2′-O-MOE), 2′-amino-DNA, 2′-fluoro-RNA, 2′-F-ANA, and 2′-bridged molecules like LNA. For further examples, see, e.g., Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3 (2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Scheme 1 below shows illustrations of some 2′-substituted modified nucleosides.

Locked Nucleic Acids (LNA)

An “LNA nucleoside” is a 2′-modified nucleoside which comprises a biradical linking the C2′ and C4′ of the ribose sugar ring of said nucleoside (also referred to as a “2′-4′ bridge”), which restricts or locks the conformation of the ribose ring. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. The locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.

Non-limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic & Med. Chem. Lett. 2002, 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75 (5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37 (4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

Further non limiting, exemplary LNA nucleosides are disclosed in Scheme 2.

Particular LNA nucleosides are beta-D-oxy-LNA, 6′-methyl-beta-D-oxy LNA such as(S)-6′-methyl-beta-D-oxy-LNA (ScET) and ENA. A particularly advantageous LNA is beta-D-oxy-LNA.

Internucleoside Linkages

The term “internucleoside linkage” is defined, as generally understood by the skilled person, as a linkage that covalently couples two nucleosides together. In antisense oligonucleotides as described herein, internucleoside linkages covalently couple adjacent nucleosides together, typically forming a bond between the sugar moieties of the adjacent nucleosides. Non-limiting examples of internucleoside linkages include phosphodiester (PO) linkages and modified internucleoside linkages.

Modified Internucleoside Linkages

The term “modified internucleoside linkage” is defined as generally understood by the skilled person as a linkage other than a phosphodiester (PO) linkage that covalently couples two nucleosides together. Modified internucleoside linkages may increase the nuclease resistance of the oligonucleotide compared to a phosphodiester (PO) linkage. Modified internucleoside linkages can stabilize oligonucleotides for in vivo use and may protect against nuclease cleavage at regions of DNA or RNA nucleosides in an oligonucleotide, for example within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides, such as region F and F′.

Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g., snake venom phosphodiesterase (SVPD)), both are well known in the art. In some oligonucleotides, all of the internucleoside linkages of an oligonucleotide, or contiguous nucleotide sequence thereof, may be nuclease resistant internucleoside linkages. It is contemplated that nucleosides, which link an oligonucleotide to a non-nucleotide functional group, such as a conjugate, may be phosphodiester.

Phosphorothioate Internucleoside Linkages

A preferred modified internucleoside linkage is phosphorothioate (PS). Phosphorothioate internucleoside linkages can be useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some oligonucleotides, all modified internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate linkages.

Nuclease resistant linkages, such as phosphorothioate linkages, can, for example, be useful in oligonucleotide regions capable of recruiting nuclease when forming a duplex with the target nucleic acid, such as region G for gapmers. Phosphorothioate linkages may, however, also be useful in non-RNase H recruiting regions and/or affinity enhancing regions such as regions F and F′ for gapmers.

Non-bridging phosphorodithioate internucleoside linkages

Another preferred modified internucleoside linkage is a non-bridging phosphorodithioate internucleoside linkage (PS₂). A PS₂ internucleoside linkage has two identical sulphur atoms attached to the phosphorous atom, achieved by replacing the non-bridging oxygen atom in the phosphorothioate linkage with a sulphur atom. Phosphorodithioate internucleoside linkages can be useful due to beneficial nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some oligonucleotides, or contiguous nucleotide sequence thereof, the most 3′ or the most 5′ internucleoside linkage is a phosphorodithioate internucleoside linkage. In some oligonucleotides, or contiguous nucleotide sequence thereof, the most 3′ and the most 5′ internucleoside linkage is a phosphorodithioate internucleoside linkage. In some oligonucleotides, all modified internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorodithioate linkages.

Nuclease resistant linkages, such as phosphorodithioate linkages, can, for example, be useful in oligonucleotide regions capable of recruiting nuclease when forming a duplex with the target nucleic acid, such as region G for gapmers. Phosphorodithioate linkages may, however, also be useful in non-RNase H recruiting regions and/or affinity enhancing regions such as regions F and F′ for gapmers.

Complementarity

The term “complementarity” describes the capacity for Watson-Crick base-pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U). It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-pairing between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

The term “% complementary” as used herein, refers to the number of nucleotides in percent of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., oligonucleotide) which, at a given position, are complementary to (i.e., form Watson Crick base pairs with) a contiguous sequence of nucleotides, at a given position of a separate nucleic acid molecule (e.g., the target nucleic acid or target sequence). The percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with the target sequence 5′-3′ and the oligonucleotide sequence from 3′-5′), dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch. Preferably, insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence.

The term “fully complementary”, refers to 100% complementarity.

Identity

The term “identity” as used herein, refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., oligonucleotide) which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g., a sequence motif). The percentage of identity is thus calculated by counting the number of aligned bases that are identical (a match) between two sequences (e.g., in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence), dividing that number by the total number of nucleotides in the aligned region and multiplying by 100. Therefore, Percentage of Identity=(Matches×100)/Length of aligned region (e.g., the contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation of the percentage of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g., 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

Hybridization

The term “hybridizing” or “hybridizes” as used herein is to be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T_m) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T_m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° is a more accurate representation of binding affinity and is related to the dissociation constant (K_d) of the reaction by ΔG°=−RTIn(K_d), where R is the gas constant and T is the absolute temperature. Therefore, a very low ΔG° of the reaction between an oligonucleotide and the target nucleic acid reflects a strong hybridization between the oligonucleotide and target nucleic acid. ΔG° is the energy associated with a reaction where aqueous concentrations are 1M, the pH is 7, and the temperature is 37° C. The hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions ΔG° is less than zero. ΔG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for ΔG° measurements. ΔG° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. In order to have the possibility of modulating its intended nucleic acid target by hybridization, oligonucleotides of the present invention hybridize to a target nucleic acid with estimated ΔG° values below −10 kcal for oligonucleotides that are 10-30 nucleotides in length. For example, the degree or strength of hybridization can be measured by the standard state Gibbs free energy ΔG°. The oligonucleotides may hybridize to a target nucleic acid with estimated ΔG° values below the range of −10 kcal, such as below −15 kcal, such as below −20 kcal and such as below −25 kcal for oligonucleotides that are 8-30 nucleotides in length. The oligonucleotides may, for example, hybridize to a target nucleic acid with an estimated ΔG° value of −10 to −60 kcal, such as −12 to −40, such as from −15 to −30 kcal or −16 to −27 kcal such as −18 to −25 kcal.

Target Nucleic Acid

A target nucleic acid is a nucleic acid to which an antisense oligonucleotide can hybridize and thereby modulate the expression of a target gene. The target nucleic acid can, for example, be a gene, an RNA, an mRNA, a pre-mRNA, a long non-coding RNA (lncRNA), a mature mRNA or a cDNA sequence or a synthetic nucleic acid derived from DNA or RNA. A target nucleic acid which is an RNA can be referred to as an “RNA target sequence”, a “target RNA sequence” or the like.

Target Sequence

The term “target sequence” as used herein refers to a sequence of nucleotides present in the target nucleic acid, which comprises the nucleobase sequence, which is complementary to an antisense oligonucleotide as described herein. The target sequence may, for example, consist of a region on the target nucleic acid, which is complementary to the contiguous nucleotide sequence of the oligonucleotide of the invention.

Target Cell

The term a “target cell” as used herein refers to a cell, which is expressing the target nucleic acid. Suitably, the target cell comprises at least one copy of the target gene in its genome. The target cell may be in vivo or in vitro. The target cell may, for example, be a mammalian cell such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell such as a monkey cell (e.g., a cynomolgus monkey cell) or a human cell.

Modulation of Expression

The term “modulation of expression” as used herein is to be understood as an overall term for an oligonucleotide's ability to alter the amount of protein expressed or RNA transcribed from the target gene. Modulation of expression may be determined by reference to a control experiment. The control may be an individual or target cell treated with a saline composition or an individual or target cell treated with a non-targeting oligonucleotide (mock).

High Affinity Modified Nucleosides

A high affinity modified nucleoside is a modified nucleotide which, when incorporated into the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (T^m). A high affinity modified nucleoside preferably results in an increase in melting temperature between +0.5 to +12° C., more preferably between +1.5 to +10° C. and most preferably between +3 to +8° C. per modified nucleoside. Numerous high-affinity modified nucleosides are known in the art and include for example, many 2′-sugar substituted nucleosides such as 2′-O-MOE, 2′-F-RNA, and LNA and analogs thereof (see e.g., Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3 (2), 293-213).

RNase H Activity and Recruitment

The ribonuclease (RNase) H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO 01/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. Recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland. Typically, an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial cleavage rate of target RNA molecules, as measured in pmol/l/min, which is at least 5%, such as at least 10% or more than 20% of the initial cleavage rate determined when using a oligonucleotide having the same base sequence as the antisense oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Example 91-95 of WO 01/23613 (hereby incorporated by reference).

Gapmer

The antisense oligonucleotide, or contiguous nucleotide sequence thereof, may be or comprise a gapmer. Gapmers are commonly used to inhibit a target nucleic acid via RNase H mediated degradation. A gapmer comprises at least three distinct structural regions-a 5′-flank, a gap and a 3′-flank-in the ‘5→3’ orientation, herein represented as 5′-F-G-F′-3′ (Formula I). The “gap” region (G) comprises a stretch of contiguous DNA nucleotides, which enable the oligonucleotide to recruit RNase H. The gap region is flanked by a 5′ flanking region (F) comprising one or more sugar-modified nucleosides, and by a 3′ flanking region (F′) comprising one or more sugar-modified nucleosides. The one or more sugar-modified nucleosides in region F and F′ may enhance the affinity of the oligonucleotide for the target nucleic acid (i.e., are affinity enhancing sugar-modified nucleosides, such as high-affinity modified nucleosides) or may modulate other properties as desired.

In a gapmer design, the 5′- and 3′-most nucleosides of the gap region are typically DNA nucleosides and are positioned adjacent to a sugar-modified nucleoside of the 5′ (F) or 3′ (F′) region respectively. The flanks may further be defined by having at least one sugar modified nucleoside at the end most distant from the gap region, i.e., at the 5′ end of the 5′ flank and at the 3′ end of the 3′ flank.

Regions F-G-F′ form a contiguous nucleotide sequence. An antisense oligonucleotide, or a contiguous nucleotide sequence thereof, may comprise or consist of a gapmer of formula I, i.e., F-G-F′.

The overall length of the gapmer design F-G-F′ is typically from 12 to 32 nucleosides, such as from 12 to 28, such as from 12 to 26, such as from 14 to 26, such as from 14 to 24, such as from 14 to 22, such as from 16 to 22 nucleosides, such as from 16 to 20 nucleosides.

Traditional gapmer designs, which do not comprise TNA nucleosides, include, for example, F_1-8-G_5-18-F′_1-8 (II), such as F_1-8-G_7-16-F′_2-8 (III), typically with the proviso that the overall length of the gapmer regions F-G-F′ is at least 12, such as at least 14 nucleotides in length.

Designs suitable for TNA (PO) gapmers according to the present invention include, for example, F_1-15-G_3-18-F′_1-15 (IV), typically with the proviso that the overall length of the gapmer regions F-G-F′ is at least 12, such as at least 14 nucleotides in length. Additional designs (e.g., Formulas IV to VII) for such gapmers are described in more details elsewhere herein.

Regions F, G and F′ are further described below and can be incorporated into any of the F-G-F′ formulae.

Gapmer-Region G

Region G (gap region) of the gapmer is a region of nucleosides, which enables the oligonucleotide to recruit RNase H, such as human RNase H1, typically DNA nucleosides. RNaseH is a cellular enzyme, which recognizes the duplex between DNA and RNA, and enzymatically cleaves the RNA molecule.

Traditional gapmers, which do not comprise TNA nucleosides, include, for example, a gap region (G) of at least 5 or 6 contiguous DNA nucleosides, such as 5-16 contiguous DNA nucleosides, such as 6-15 contiguous DNA nucleosides, such as 7-14 contiguous DNA nucleosides, such as 8-12 contiguous DNA nucleotides, such as 8-12 contiguous DNA nucleotides in length.

Suitable gapmers according to the present invention, particularly gapmers comprising one or more TNA (PO) nucleosides as described herein, may have a gap region (G) comprising at least 3 contiguous DNA nucleosides. The gap region G may, for example, comprise or consist of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 contiguous DNA nucleosides. Preferably, the gap region G comprises at least 4, at least 5, or at least 6 contiguous DNA nucleosides.

A gap region (G) which comprises one or more TNA (PO) nucleosides as described herein has a DNA nucleoside at the 5′ end of the gap (adjacent to the 3′ nucleoside of region F), and a DNA nucleoside at the 3′ end of the gap (adjacent to the 5′ nucleoside of region F′), typically retaining a region of at least 3 or 4 contiguous DNA nucleosides at either the 5′ end, the 3′ end, or both, of the gap region.

The total length of a gap region G is typically up to 18 contiguous nucleosides. For example, the total length of the gap region G may be from 3 to 18 contiguous nucleosides, such as from 3 to 16 contiguous nucleosides, such as from 4 to 18, 4 to 16, 4 to 14, 4 to 12, or 4 to 10 contiguous nucleosides, such as from 5 to 18, 5 to 16, 5 to 14, 5 to 12, or 5 to 10 contiguous nucleosides, such as from 6 to 18, 6 to 16, 6 to 14, 6 to 12, or 6 to 10 contiguous nucleosides. Shorter gap regions are also contemplated, such as a region G comprising or consisting of 4, 5, 6, 7, 8 or 9 contiguous nucleosides, e.g., contiguous DNA nucleosides.

One or more cytosine (C) DNA nucleosides in the gap region may in some instances be methylated (e.g., when a C DNA nucleoside is followed by a guanine (G) DNA nucleoside and annotated as 5-methyl-cytosine (^meC or ^mC).

Oligonucleotides contemplated include those where all the internucleoside linkages in the gap are phosphorothioate linkages, or where all modified internucleoside linkages in the gap are phosphorothioate linkages.

Whilst traditional gapmers have a DNA gap region, there are numerous examples of modified nucleosides, which allow for RNase H recruitment when they are used within the gap region. Modified nucleosides which have been reported as being capable of recruiting RNase H when included within a gap region include, for example, alpha-L-LNA, C4′ alkylated DNA (as described in PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, both incorporated herein by reference), arabinose derived nucleosides like ANA and 2′F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661), UNA (unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst., 2009, 10, 1039 incorporated herein by reference). UNA or “unlocked nucleic acid” is typically where the bond between C2 and C3 of the ribose has been removed, forming an unlocked “sugar” residue. The modified nucleosides used in gapmers may be nucleosides which adopt a 2′-endo (DNA-like) structure when introduced into the gap region, allowing for RNaseH recruitment. The DNA Gap region (G) described herein may, for example, optionally contain 1 or more (e.g., 1 to 3) sugar modified nucleosides. Any two or more sugar-modified nucleosides in the gap may be consecutive or separated by one or more DNA nucleosides.

As described herein, modified nucleosides which may be used in the gap region include TNA (PO) nucleosides.

Region G—“Gap-breaker”

There are also numerous reports on the insertion of modified nucleosides, which confer a 3′ endo conformation into the gap region of gapmers, whilst retaining some RnaseH activity. Gapmers with a gap region comprising one or more 3′ endo modified nucleosides are referred to as “gap-breaker” or “gap-disrupted” gapmers, see for example WO2013/022984. Gap-breaker oligonucleotides retain sufficient region of DNA nucleosides within the gap region to allow for RnaseH recruitment. The ability of gap-breaker oligonucleotide design to recruit RnaseH is typically sequence or even compound specific—see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487, which discloses “gap-breaker” oligonucleotides, which recruit RnaseH which in some instances provide a more specific cleavage of the target RNA. Modified nucleosides used within the gap region of gap-breaker oligonucleotides may for example be modified nucleosides which confer a 3′-endo conformation, such 2′-O-methyl (Ome) or 2′-O-MOE (MOE) nucleosides, or beta-D LNA nucleosides (the bridge between C2′ and C4′ of the ribose sugar ring of a nucleoside is in the beta conformation), such as beta-D-oxy LNA or ScET nucleosides.

TNA (PO) nucleosides may also be contemplated as gap-breakers.

As with gapmers containing region G described above, the gap region of gap-breaker or gap-disrupted gapmers, have a DNA nucleoside at the 5′ end of the gap (adjacent to the 3′ nucleoside of region F), and a DNA nucleoside at the 3′ end of the gap (adjacent to the 5′ nucleoside of region F′). Gapmers which comprise a disrupted gap typically retain a region of at least 3 or 4 contiguous DNA nucleosides at either the 5′ end, the 3′ end, or both, of the gap region.

Exemplary designs for gap-breaker gapmers as described herein include

F 1 ⁢ ‐ ⁢ 15 ⁢ ‐ [ D 3 ⁢ ‐ ⁢ 4 ‐ E 1 ‐ D 3 ⁢ ‐ ⁢ 4 ] ‐ F 1 ⁢ ‐ ⁢ 15 ′ F 1 ⁢ ‐ ⁢ 15 ⁢ ‐ [ D 1 ⁢ ‐ ⁢ 4 ‐ E 1 ‐ D 3 ⁢ ‐ ⁢ 4 ] ‐ F 1 ⁢ ‐ ⁢ 15 ′ F 1 ⁢ ‐ ⁢ 15 ⁢ ‐ ⁢ [ D 3 ⁢ ‐ ⁢ 4 ‐ E 1 ‐ D 1 ⁢ ‐ ⁢ 4 ] ‐ F 1 ⁢ ‐ ⁢ 15 ′

wherein region G is within the brackets [D_n-E_r-D_m], D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (a gap-breaker or gap-disrupting nucleoside), and F and F′ are the flanking regions as defined herein, and with the proviso that the overall length of the gapmer regions F-G-F′ is at least 12, such as at least 14 nucleotides in length.

Region G of a gap disrupted gapmer as described herein may comprise at least 4 DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 DNA nucleosides. As described above, the DNA nucleosides may be contiguous or may optionally be interspersed with one or more modified nucleosides, with the proviso that the gap region G is capable of mediating RNase H recruitment.

Gapmer—Flanking Regions, F and F′

Region F is positioned immediately adjacent to the 5′ DNA nucleoside of region G. The 3′ most nucleoside of region F is a sugar modified nucleoside. Advantageously the one or two 5′ most nucleosides of region F are also sugar modified nucleosides. In a gapmer as described herein, particularly a gapmer comprising one or more TNA (PO) nucleosides, Region F is at least one, such as at least 2, such as at least 3 contiguous nucleotides in length. Typically, region F is up to 15 contiguous nucleotides in length. For example, Region F can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous nucleotides in length, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 contiguous nucleotides in length.

Region F′ is positioned immediately adjacent to the 3′ DNA nucleoside of region G. The 5′ most nucleoside of region F′ is a sugar modified nucleoside. Advantageously the one or two 3′ most nucleosides of region F′ are also sugar modified nucleosides. In a gapmer as described herein, particularly a gapmer comprising one or more TNA (PO) nucleosides, Region F′ is at least one, such as at least 2, such as at least 3 contiguous nucleotides in length. Typically, region F′ is up to 15 contiguous nucleotides in length. For example, Region F′ can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous nucleotides in length, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 contiguous nucleotides in length.

Any sugar-modified nucleotide can be used in region F and/or F′ of an antisense oligonucleotide as described herein provided that the antisense oligonucleotide retains the capability to recruit RNase H and any other desired properties. Examples of sugar-modified nucleosides for use in F, F′, or both of F and F′, are described herein and include, without limitation, those disclosed in the sections entitled “Sugar-modified nucleosides,” including those more particularly described in the sections entitled “Threose nucleic acids (TNA), “2′-Sugar-modified nucleosides” and “Locked nucleic acids.” A TNA (PO) gapmer as described herein may, for example, comprise one or more TNA nucleosides, LNA nucleosides, MOE nucleosides, or mixtures thereof.

LNA Gapmer

An LNA gapmer is a gapmer wherein one or both of region F and F′ comprise or consist of LNA nucleosides. A beta-D-oxy LNA gapmer is a gapmer wherein either one or both of region F and F′ comprises or consists of beta-D-oxy LNA nucleosides. An LNA gapmer can, for example, have the formula: [LNA]_1-5-[region G]-[LNA]_1-5, wherein region G is as described in the section entitled “Gapmer-Region G.” An example of a specific LNA gapmer design is 3-10-3 (LNA-DNA-LNA).

cEt Gapmer

A cEt gapmer is a gapmer wherein one or both of region F and F′ comprise or consist of cEt nucleosides. A beta-D-oxy cEt gapmer is a gapmer wherein either one or both of region F and F′ comprises or consists of beta-D-oxy cEt nucleosides. A cEt gapmer can, for example, have the formula: [cEt]_1-5-[region G]-[cEt]_1-5, wherein region G is as described in the section entitled “Gapmer-Region G.” An example of a specific cEt gapmer design is 3-10-3 (cEt-DNA-cEt).

MOE Gapmer

A MOE gapmer is a gapmer wherein one or both of regions F and F′ comprise or consist of MOE nucleosides, e.g., 2′-O-MOE nucleosides. A MOE gapmer can, for example, have the formula [MOE]_1-8-[Region G]-[MOE]_1-8, such as [MOE]_2-7-[Region G]_5-16-[MOE]_2-7, such as [MOE]_3-6-[Region G]-[MOE]_3-6, wherein region G is as described in the section entitled “Gapmer-Region G”. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) have been widely used in the art.

TNA Gapmer

A “TNA gapmer” or “TNA modified gapmer” is a gapmer wherein the linked nucleosides of one or more of regions F, F′ and G comprise at least one TNA nucleoside.

A TNA gapmer can, for example, have a formula in which the nucleosides of F, F′, or both F and F′, consist of TNA nucleosides.

TNA (PO) Gapmer

A “TNA (PO) gapmer” or “TNA (PO) modified gapmer” is a gapmer wherein the linked nucleosides of one or more of regions F, F′ and G comprise at least one TNA nucleoside linked to at least one adjacent nucleoside by a phosphodiester (PO) internucleoside linkage.

A TNA (PO) gapmer can, for example, have a formula in which the nucleosides of F, F′, or both F and F′, consist of TNA nucleosides linked by PO internucleoside linkages. These and other examples of specific designs of TNA (PO) gapmers are described elsewhere herein.

Mixed Wing Gapmer

A mixed wing gapmer is a gapmer wherein one or both of region F and region F′ comprise more than one type of sugar-modified nucleosides. Many sugar-modified nucleosides are known in the art and contemplated for this purpose. The two or more different sugar-modified nucleosides in a flank region can, for example, be selected from the those disclosed in the sections entitled “Sugar-modified nucleosides,” including but limited to those disclosed in the sections entitled “Threose nucleic acids (TNA)”, “2′-Sugar-modified nucleosides” and “Locked nucleic acids.”

Mixed-wing gapmers contemplated include, for example, those where at least one of region F and region F′ comprises a TNA or TNA (PO) nucleoside. The other sugar modified nucleoside(s) may then be selected from, for example, a 2′ substituted nucleoside, such as a 2′ substituted nucleoside independently selected from the group consisting of 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNA units, 2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, arabino nucleic acid (ANA) units and 2′-fluoro-ANA units, such as MOE nucleosides.

Also contemplated are mixed wing gapmers wherein, when at least one of region F and F′, or both region F and F′ comprise at least one TNA or TNA (PO) nucleoside, the remaining nucleosides of region F and F′ are independently selected from the group consisting of MOE and LNA. When at least one of region F and F′, or both region F and F′ comprise at least two LNA nucleosides, the remaining nucleosides of region F and F′ can, for example, be independently selected from the group consisting of MOE and LNA. In some mixed wing gapmers, one or both of region F and F′ may further comprise one or more DNA nucleosides.

Mixed wing gapmer designs are disclosed in WO2008/049085 and WO2012/109395, both of which are hereby incorporated by reference.

Alternating Flank Gapmer

Oligonucleotides with alternating flanks are gapmer oligonucleotides where at least one of the flanks (F or F′) comprises DNA in addition to a sugar modified nucleoside, e.g., a sugar-modified nucleoside selected from those described herein in the sections entitled “Sugar-modified nucleosides,” including but limited to those described in the sections entitled “Threose nucleic acids”, “2′-Sugar-modified nucleosides” and “Locked nucleic acids.” For example, apart from DNA, an alternating flank gapmer may comprise TNA, TNA (PO), LNA and/or MOE nucleoside(s).

For example, at least one of region For F′, or both region F and F′, may comprise both sugar modified nucleosides and DNA nucleosides. The flanking region For F′, or both F and F′, typically then comprises at least three nucleosides, wherein the 5′ and 3′ most nucleosides of the F and/or F′ region are sugar-modified nucleosides.

Region D′ or D″

Antisense oligonucleotides as described herein may comprise further 5′ and/or 3′ nucleosides which are not fully complementary to the target nucleic acid. The further 5′ and/or 3′ nucleosides may be referred to as region D′ and D″ herein.

The addition of region D′ or D″ may be used for the purpose of joining the contiguous nucleotide sequence, such as the gapmer, to a conjugate moiety or another functional group. When used for joining the contiguous nucleotide sequence with a conjugate moiety can serve as a cleavable linker. It may also or alternatively be used to provide exonuclease protection or for ease of synthesis or manufacture.

Region D′ and D″ can be attached to the 5′ end of region For the 3′ end of region F′, respectively to generate designs of the following formulas D′-F-G-F′, F-G-F′-D″ or D′-F-G-F′-D″. In this instance the F-G-F′ is the gapmer portion of the oligonucleotide and region D′ or D″ constitute a separate part of the oligonucleotide.

Region D′ or D″ may independently comprise or consist of 1, 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid. The nucleotide adjacent to the F or F′ region is not a sugar-modified nucleotide, such as a DNA or RNA or base modified versions of these. The D′ or D′ region may serve as a nuclease susceptible bio-cleavable linker. For example, the additional 5′ and/or 3′ end nucleotides can be DNA or RNA nucleotides and can be linked by phosphodiester linkages.

Nucleotide based bio-cleavable linkers suitable for use as region D′ or D″ are disclosed in WO2014/076195, which include by way of example a phosphodiester linked DNA dinucleotide. The use of bio-cleavable linkers in poly-oligonucleotide constructs is disclosed in WO2015/113922, where they are used to link multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

Conjugates

The term “conjugate” as used herein refers to an oligonucleotide, which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).

Conjugation of antisense oligonucleotides described herein to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, e.g., by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. Conjugation may, for example, modify or enhance the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In particular the conjugate may target the oligonucleotide to a specific organ, tissue or cell type and thereby enhance the effectiveness of the oligonucleotide in that organ, tissue or cell type. At the same time the conjugate may serve to reduce activity of the oligonucleotide in non-target cell types, tissues or organs, e.g., off target activity or activity in non-target cell types, tissues or organs.

The non-nucleotide moiety (conjugate moiety) can, for example, be selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids) or combinations thereof.

Linkers

A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. Conjugate moieties can be attached to the oligonucleotide directly or through a linking moiety (e.g., linker or tether). Linkers serve to covalently connect a third region, e.g., a conjugate moiety (Region C), to a first region, e.g., an oligonucleotide or contiguous nucleotide sequence or gapmer region F-G-F′ (region A).

A conjugate or oligonucleotide conjugate may optionally comprise a linker region (second region or region B and/or region Y) which is positioned between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).

Region B refers to bio-cleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. Conditions under which physiologically labile linkers undergo chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases. The bio-cleavable linker may, for example, be susceptible to S1 nuclease cleavage. DNA phosphodiester containing bio-cleavable linkers are described in more detail in WO 2014/076195 (hereby incorporated by reference)—see also region D′ or D″ herein.

Region Y refers to linkers that are not necessarily bio-cleavable but primarily serve to covalently connect a conjugate moiety (region C or third region), to an oligonucleotide (region A or first region). The region Y linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups. The oligonucleotide conjugates can be constructed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. The linker (region Y) can, for example, be an amino alkyl, such as a C₂-C₃₆ amino alkyl group, including, for example C₆ to C₁₂ amino alkyl groups. Preferably, the linker (region Y) is a C6 amino alkyl group.

Treatment

The term ‘treatment’ as used herein refers to both treatment of an existing disease (e.g., a disease or disorder as herein referred to), or prevention of a disease, i.e., prophylaxis. It will therefore be recognized that treatment as referred to herein may be prophylactic.

TNA-Modified Antisense Oligonucleotides

Despite being non-natural, Threose Nucleic Acids (TNAs) can form stable Watson-Crick duplexes and show strong affinity and specificity toward complementary RNA targets. As shown herein, TNA (PO) modified gapmers provide new design strategies for antisense oligonucleotide applications, particularly as an alternative to traditional gapmers employing only PS linkages. Using Caspase 3/7 activation, in vitro target knockdown and thermal melting assays, it was demonstrated that TNA (PO) modification can be used to mitigate toxicity while still maintaining target knockdown efficacy and affinity for the target nucleic acid. For example, in a gapmer of a state-of-the-art design such as an LNA or MOE gapmer, TNA (PO) units can replace one or more or all LNA or MOE units in the flank regions. TNA (PO) units can also replace one or more or up to all but three or four consecutive DNA units in the gap region of a gapmer of a state-of-the-art design, for example, which may effectively result in an extended 5′ or 3′ flank and a reduced gap. Moreover, TNAs are hardly recognized by nucleases. Therefore, when designed into gapmers, TNA (PO) units can lead to maintained or increased metabolic stability, maintained or longer duration of action, or both, while reducing the sulfur content as compared to gapmer designs employing only PS linkages. TNA (PO) modified gapmers can therefore yield long-acting therapeutic agents with an increased therapeutic index compared to classical gapmer designs.

Accordingly, the invention provides an antisense oligonucleotide, such as an antisense gapmer oligonucleotide, comprising a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage. The TNA nucleoside can be linked to an adjacent nucleoside by a 2′-PO internucleoside linkage or a 3′-PO internucleoside linkage. The TNA nucleoside can also be linked to any adjacent nucleoside by a 2′-PO or 3′-PO internucleoside linkage. Non-limiting examples of adjacent nucleosides include sugar-modified nucleosides and DNA nucleosides. The TNA nucleoside can, for example, be linked to a first and a second adjacent nucleoside by a 2′-PO internucleoside linkage and a 3′-modified internucleoside linkage, respectively;

• • a 2′-modified internucleoside linkage and a 3′-PO internucleoside linkage, respectively; or
  • a 2′-PO internucleoside linkage and a 3′-PO internucleoside linkage, respectively,
  • optionally wherein any 2′-modified or 3′-modified internucleoside linkage is a PS internucleoside linkage, or wherein any 2′-modified or 3′-modified internucleoside linkage is a PS₂ internucleoside linkage.

The invention also provides an antisense gapmer oligonucleotide, such as an antisense gapmer oligonucleotide, comprising one or more TNA (PO) nucleotides, typically selected from the group consisting of 2′-PO linked, 3′-PO linked and 2′,3′-PO linked TNA nucleosides.

The antisense gapmer oligonucleotide may particularly comprise a contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I) which is capable of recruiting ribonuclease (RNase) H. A contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I) which comprises at least one TNA (PO) nucleoside can be referred to herein as a “TNA (PO) gapmer”. Contemplated designs for a TNA (PO) gapmer include those wherein

• • G is a gap region of up to 18 linked nucleosides which comprises at least 3 contiguous DNA nucleosides,
  • each of F and F′ is a flanking region of up to 15 linked nucleosides which independently comprises or consists of 1 to 15 sugar-modified nucleosides, and
  • at least one of F, F′ and G comprises a sugar-modified nucleoside which is a TNA (PO) nucleoside.

While the following sections provide more details on TNA (PO) gapmers, it should be understood that, unless otherwise indicated or contradicted by context, they apply equally to an antisense gapmer oligonucleotide, or a conjugate thereof, which comprises or consist of a TNA (PO) gapmer.

Advantageously, a TNA (PO) gapmer can modulate the expression of a target gene by reducing or inhibiting its expression into mRNA and/or a protein, typically by hybridizing to a target nucleic acid. When the target nucleic acid is an RNA, e.g., a pre-mRNA, mRNA, viral RNA, microRNA or lncRNA target nucleic acid, the TNA (PO) gapmer is capable of reducing or inhibiting the expression of the target RNA. This is achieved by the complementarity between the TNA (PO) gapmer and the target RNA, and, suitably, the recruitment of a cellular RNase such as RNase H. The TNA (PO) gapmer may additionally be able to reduce or inhibit the expression of a target RNA by non-RNase H mediated mechanisms, such as a steric blocking mechanism resulting in microRNA inhibition, reduced splice modulation of pre-mRNAs, or blocking of the interaction between an lncRNA and chromatin.

Preferably, the TNA (PO) gapmer can reduce the expression level of the target by at least about 20% compared to the normal expression level of the target, more preferably by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the normal expression level of the target. The TNA (PO) gapmer can preferably also or alternatively inhibit the expression of the target by at least about 20% compared to the normal expression level of the target, more preferably by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the normal expression level of the target.

Assays for evaluating the reduction of the expression level or inhibition of expression of a particular target are known to the skilled person. Suitable assays include in vitro assays using target cells which comprise at least one copy of the target gene in the genome and express the target, e.g., the target RNA. For example, in an in vitro assay where target cells are incubated with about 25 μM TNA (PO) gapmer, a TNA (PO) gapmer can be capable of reducing the expression levels of an RNA target by at least about 50%, such as at least about 60%, as compared to the normal expression level of the RNA target. In such an assay, a TNA (PO) gapmer can also or alternatively be capable of inhibiting the expression of an RNA target by at least about 50%, such as at least about 60%, as compared to the normal expression of the RNA target. At a concentration of about 25 μM, a TNA (PO) gapmer may also be able to reduce or inhibit the expression level of an RNA target by at least about 70%, such as at least about 80%, such as at least about 90% as compared to the normal expression level of the target.

The normal expression level of the RNA target can be determined using a control where the target cells are incubated without TNA (PO) gapmer (e.g., in the presence of vehicle only) or with an irrelevant control oligonucleotide. Target cells for in vitro assays can be obtained from commercial sources (e.g., in the form of cell lines) or isolated from blood or other tissues of a human or an experimental animal. The target cells may, for example, be incubated with the TNA (PO) gapmer or control for about 1 to 5 days, such as about 2, 3 or 4 days, such as about 3 days. The RNA can then be extracted and the level of remaining target RNA in test and control samples determined by gene expression analysis. Alternatively, instead of determining the remaining target RNA, the level of an RNA species (e.g., mRNA) or protein derived from the target RNA can be determined in test and control samples. An example of a typical assay for evaluating reduction of the expression level or inhibition of expression of a target RNA by a TNA (PO) gapmer, which can be adapted to other targets and target cells, is provided in Example 2.

The ability of a TNA (PO) gapmer to reduce the expression level of the target or inhibit the expression of the target can also be evaluated by determining the IC50 value, i.e., the concentration of the TNA (PO) gapmer where the expression level of the target nucleic acid is reduced by half. In an in vitro assay using target cells which comprise at least one copy of the target gene in the genome and express the target, e.g., the target RNA, the IC50 is preferably no more than about 20 μM, such as no more than about 10 μM, such as no more than about 5 μM. Typically, the IC50 value is determined in a cell assay similar to that already described except that target cells are incubated with a dilution series of the TNA (PO) gapmer which spans the IC50 value. An example of a typical assay for evaluating the IC50 reduction of the expression level or inhibition of expression of a target RNA by a TNA (PO) gapmer, which can be adapted to other targets and target cells, is provided in Example 3.

The ability of a TNA (PO) gapmer can also be evaluated in relation to a control or “parent” gapmer from which the TNA (PO) gapmer is derived and which does not comprise any TNA nucleoside. The IC50 value of a TNA (PO) gapmer is preferably no more than about 10, no more than about 8, no more than about 6, no more than about 4, or no more than about 2 times that of the control or “parent” gapmer.

A TNA (PO) gapmer can also be characterized by having a low toxicity. For example, the TNA (PO) gapmer may have a lower toxicity than a corresponding control gapmer, such as a state-of-the-art reference gapmer or “parent” gapmer which differs from the TNA (PO) gapmer in that its nucleosides do not include any TNA nucleoside. Suitable assays for evaluating the toxicity of a gapmer or antisense nucleotide are known in the art and include, for example, in vitro assays such as Caspase 3/7 assays. Caspase 3/7 assays reflect the level of apoptosis induced by a compound and are suitable for, e.g., evaluating the risk for hepatotoxicity of a compound based on tests on liver cells or cell lines. Briefly, HepG2 cells from a commercial source can be transfected with 100 nM TNA (PO) or control gapmers in a suitable vehicle and Caspase 3/7 activation determined at about 24 hours post-transfection. Preferably, the Caspase 3/7 activation from transfection with a TNA (PO) gapmer is at most about 70%, such as at most about 60%, such as at most about 50%, such as at most about 40%, such as at most about 30%, such as at most about 20% of the corresponding control gapmer. Alternatively, using the Caspase 3/7 assay described in Example 4, the percentage determined (% assay window, reflecting apoptotic cells over the total cells), for a TNA (PO) gapmer is preferably at most about 250%, more preferably at most about 200%, more preferably at most about 150%, at most about 100%, at most about 80%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or at most about 10%. Preferably, using the using the Caspase 3/7 assay described in Example 4, the percentage determined (% assay window) for a TNA (PO) gapmer is at most about 80%, such as at most about 60%.

A TNA (PO) gapmer is capable of hybridizing to the target nucleic acid, e.g., a target RNA, particularly to the target sequence to which its nucleobase sequence is complementary. The ability of a TNA (PO) gapmer to hybridize to its target nucleic acid can be evaluated according to any assay known in the art. Advantageously, thermal melting (Tm) analysis can be used, determining at which temperature a duplex between a TNA (PO) gapmer and its RNA target sequence denatures, which can be denoted the melting temperature or simply Tm. A typical assay for determining the Tm of a TNA (PO) gapmer (i.e., in the form of a duplex with a complementary RNA target sequence) is described in Example 5. Briefly, TNA (PO) gapmer and RNA target sequence can be added to 20 mM disodium phosphate buffer, 200 mM NaCl and 0.2 mM EDTA (pH 7) resulting in a final concentration of 1.5 μM. Samples can be heated to 95° C. for 5 min and then slowly cooled to room temperature over a period of 1 hour, and thermal melting curves recorded at 260 nm using a temperature gradient, e.g., with an increase by 5° C./min from 25° C. to 95° C. and then decreased to 25° C. From the derivative of both curves, the melting temperature (Tm) can be determined. Preferably, a TNA (PO) gapmer has a Tm of at least about 50° C., such as at least about 52° C., such as at least about 54° C., such as at least about 56° C., such as at least about 58° C., such as at least about 60° C., such as at least about 65° C., such as at least about 70° C.

In some cases, there may be mismatches between the oligonucleotide and the target nucleic acid, such as 1 or 2 mismatches. Despite mismatches, the hybridization to the target nucleic acid may still be sufficient to show a desired ability to modulate the target. Preferably, a TNA (PO) gapmer

• • (a) can reduce the expression level of a target nucleic acid by at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, as compared to the normal expression level of the target;
  • (b) has an IC50 of no more than about 20 μM, such as no more than about 10 μM, such as no more than about 5 μM, for reducing the expression level of a target nucleic acid;
  • (c) using the Caspase 3/7 assay described in Example 4, the percentage assay window (% AW) is preferably at most about 60%, such as at most about 40%, such as at most about 20%, such as at most about 10%;
  • (d) has, in the form of a duplex of the antisense gapmer oligonucleotide with an RNA target sequence, a melting temperature (Tm) of at least about 50° C., such as at least about 52° C., such as at least about 54° C., such as at least about 56° C., such as at least about 58° C., such as at least about 60° C.; or
  • (e) a combination of any two or more of (a) to (d).

For example, a preferred TNA (PO) gapmer can be characterized by both features (a) and (b). Another preferred TNA (PO) gapmer can be characterized by both features (a) and (c). Another preferred TNA (PO) gapmer can be characterized by both features (a) and (d). Another preferred TNA (PO) gapmer can be characterized by both features (b) and (c). Another preferred TNA (PO) gapmer can be characterized by both features (b) and (d). Another preferred TNA (PO) gapmer can be characterized by both features (c) and (d). Another preferred TNA (PO) gapmer can be characterized by features (a), (b) and (c). Another preferred TNA (PO) gapmer can be characterized by features (a), (b) and (d). Another preferred TNA (PO) gapmer can be characterized by features (a), (c) and (c). Another preferred TNA (PO) gapmer can be characterized by features (b), (c) and (d). A preferred TNA (PO) gapmer can also be characterized by all of features (a) to (d).

Preferably, the target nucleic acid in (a) and (b) is an RNA, and the RNA target sequence in (c) has a nucleobase sequence complementary to the contiguous nucleotide sequence of the TNA (PO) gapmer. The reduction in expression level of the and (a) and (b) can, for example, be determined in a target cell expressing the target nucleic acid and incubated with the antisense gapmer oligonucleotide at a concentration of about 25 μM for about 3 days, as already described above.

In some TNA (PO) gapmers, region F comprises at least one TNA nucleoside. Region F of a TNA gapmer may, for example, comprise up to 15 TNA nucleosides.

In some TNA (PO) gapmers, region F comprises at least one TNA (PO) nucleoside. Region F may also comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve TNA (PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA (PO) nucleosides. TNA (PO) gapmers wherein region F comprises or consists of one TNA (PO) nucleoside are also contemplated.

In some TNA (PO) gapmers, region F comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA (PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen adjacent TNA (PO) nucleosides. Region F may also consist of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen adjacent TNA (PO) nucleosides.

In some TNA (PO) gapmers, at least the 3′-most nucleoside in F is a TNA nucleoside. Suitably, at least the two, at least the three, at least the four, at least the five, at least the six, at least the seven, at least the eight, at least the nine, at least the ten, at least the eleven, or at least the twelve 3′-most nucleosides in F can be TNA nucleosides. For example, the two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen 3′-most nucleosides in F can be TNA nucleosides, such as TNA (PO) nucleosides.

In some TNA (PO) gapmers, at least the 5′-most nucleoside in F is a TNA nucleoside. Suitably, at least the two, at least the three, at least the four, at least the five, at least the six, at least the seven, at least the eight, at least the nine, at least the ten, at least the eleven, or at least the twelve 5′-most nucleosides in F can be TNA nucleosides. For example, the two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen 5′-most nucleosides in F can be TNA nucleosides, such as TNA (PO) nucleosides.

In some TNA (PO) gapmers, both the 3′-most and the 5′-most nucleosides in F are TNA nucleosides. For example, the 3′-most and/or 5′-most nucleosides in F can be independently selected from one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve TNA nucleosides, such as TNA (PO) nucleosides.

Suitably, in TNA (PO) gapmers, each TNA nucleoside in F can be a TNA (PO) nucleoside. However, also contemplated are TNA (PO) gapmers, wherein, except for the 5′-most TNA nucleoside in F, each TNA nucleoside in F is a TNA (PO) nucleoside. The 5′-most TNA nucleoside can, for example, be linked to the adjacent nucleoside via a PS or PS₂ internucleoside linkage.

Any remaining nucleoside(s) in F can be one or more other sugar-modified nucleosides than a TNA (PO) nucleoside (e.g., in the form of a mixed-wing gapmer) or one or more DNA nucleosides (e.g., in the form of an alternating flank gapmer). For example, F may further comprise 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides, such as two, three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides. Non-limiting examples of sugar-modified nucleosides include those described in the section entitled “Sugar-modified nucleosides,” such as, e.g., LNA, 2′-O-MOE, and TNA nucleosides, optionally linked by PS or PS₂ internucleoside linkages.

Contemplated are also TNA (PO) gapmers wherein all sugar-modified nucleosides of F are TNA (PO) nucleosides. A TNA (PO) gapmer may, for example, be an alternating flank gapmer where the nucleosides of F consist of DNA and TNA (PO) with, e.g., 1, 2 or 3 DNA nucleosides. Suitably, at least the 5′-most and 3′-most nucleosides of F are then TNA (PO) nucleosides.

The nucleosides of F may also consist of a TNA (PO) nucleoside. Alternatively, region F may consist of more than one TNA (PO) nucleoside, such as two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA (PO) nucleosides. TNA (PO) nucleosides where the nucleosides of F consist of thirteen, fourteen or fifteen TNA (PO) nucleosides are also contemplated. Typically, when the nucleosides of F consist of two or more TNA (PO) nucleosides, F is a contiguous sequence of linked TNA (PO) nucleosides.

In some TNA gapmers, F does not comprise any TNA (PO) nucleoside.

In some TNA (PO) gapmers, region F′ comprises at least one TNA nucleoside. Region F′ of a TNA gapmer may, for example, comprise up to 15 TNA nucleosides.

In some TNA (PO) gapmers, region F′ comprises at least one TNA (PO) nucleoside. Region F′ may also comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve TNA (PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA (PO) nucleosides. TNA (PO) gapmers wherein region F′ comprises or consists of one TNA (PO) nucleoside are also contemplated.

In some TNA (PO) gapmers, region F′ comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA (PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen adjacent TNA (PO) nucleosides. Region F′ may also consist of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen adjacent TNA (PO) nucleosides.

In some TNA (PO) gapmers, at least the 5′-most nucleoside in F′ is a TNA nucleoside. Suitably, at least the two, at least the three, at least the four, at least the five, at least the six, at least the seven, at least the eight, at least the nine, at least the ten, at least the eleven, or at least the twelve 5′-most nucleosides in F′ can be TNA nucleosides. For example, the two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen 5′-most nucleosides in F′ can be TNA nucleosides, such as TNA (PO) nucleosides.

In some TNA (PO) gapmers, at least the 3′-most nucleoside in F′ is a TNA nucleoside. Suitably, at least the two, at least the three, at least the four, at least the five, at least the six, at least the seven, at least the eight, at least the nine, at least the ten, at least the eleven, or at least the twelve 3′-most nucleosides in F′ can be TNA nucleosides. For example, the two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen 3′-most nucleosides in F′ can be TNA nucleosides, such as TNA (PO) nucleosides.

In some TNA (PO) gapmers, both the 3′-most and the 5′-most nucleosides in F′ are TNA nucleosides. For example, the 3′-most and/or 5′-most nucleosides in F′ can be independently selected from one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve TNA nucleosides, such as TNA (PO) nucleosides.

Suitably, in TNA (PO) gapmers, each TNA nucleoside in F′ can be a TNA (PO) nucleoside. However, also contemplated are TNA (PO) gapmers, wherein, except for the 3′-most TNA nucleoside in F′, each TNA nucleoside in F is a TNA (PO) nucleoside. The 3′-most TNA nucleoside can, for example, be linked to the adjacent nucleoside via a PS or PS₂ internucleoside linkage.

Any remaining nucleoside(s) in F′ can be one or more other sugar-modified nucleosides than a TNA (PO) nucleoside (e.g., in the form of a mixed-wing gapmer) or one or more DNA nucleosides (e.g., in the form of an alternating flank gapmer). For example, F′ may further comprise 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides, such as two, three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides. Non-limiting examples of sugar-modified nucleosides include those described in the section entitled “Sugar-modified nucleosides,” such as, e.g., LNA, 2′-O-MOE, and TNA nucleosides, optionally linked by PS or PS₂ internucleoside linkages.

Contemplated are also TNA (PO) gapmers wherein all sugar-modified nucleosides of F′ are TNA (PO) nucleosides. A TNA (PO) gapmer may, for example, be an alternating flank gapmer where the nucleosides of F′ consist of DNA and TNA (PO) with, e.g., 1, 2 or 3 DNA nucleosides. Suitably, at least the 5′-most and 3′-most nucleosides of F′ are then TNA (PO) nucleosides.

The nucleosides of F′ may also consist of a TNA (PO) nucleoside. Alternatively, region F′ may consist of more than one TNA (PO) nucleoside, such as two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA (PO) nucleosides. TNA (PO) nucleosides where the nucleosides of F′ consist of thirteen, fourteen or fifteen TNA (PO) nucleosides are also contemplated. Typically, when the nucleosides of F′ consist of two or more TNA (PO) nucleosides, F′ is a contiguous sequence of linked TNA (PO) nucleosides.

In some TNA gapmers, F′ does not comprise any TNA nucleoside.

G comprises a stretch of contiguous DNA nucleosides which enable the antisense oligonucleotide to recruit RNase H. Suitably, G may comprise up to eighteen nucleosides, such as DNA nucleosides. Typically, at least the 5′-most nucleoside in G and the 3′-most nucleoside in G are DNA nucleosides.

In some TNA (PO) gapmers, G does not comprise any TNA nucleoside. For example, G may comprise at least four DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous DNA nucleosides.

In some TNA (PO) gapmers according to the present invention, G comprises at least one TNA nucleoside. For example, depending on the overall length of region G, the second, third, fourth, fifth, sixth, seventh-most, or eight-most 5′ nucleoside in G can be a TNA nucleoside. Alternatively, depending on the overall length of region G, the second, third, fourth, fifth, sixth, seventh-most, or eight-most 3′ nucleoside in G can be a TNA nucleoside.

Also contemplated are TNA (PO) gapmers where G comprises at least two or at least three TNA nucleosides. The at least two or at least three TNA nucleosides may be consecutive or non-consecutive. Region G may, for example, comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen TNA nucleosides, optionally consecutive.

In some TNA (PO) gapmers according to the present invention, G comprises at least one TNA (PO) nucleoside. For example, depending on the overall length of region G, the second, third, fourth, fifth, sixth, seventh-most, or eight-most 5′ nucleoside in G can be a TNA (PO) nucleoside. Alternatively, depending on the overall length of region G, the second, third, fourth, fifth, sixth, seventh-most, or eight-most 3′ nucleoside in G can be a TNA nucleoside.

Also contemplated are TNA (PO) gapmers where G comprises at least two or at least three TNA (PO) nucleosides. The at least two or at least three TNA (PO) nucleosides may be consecutive or non-consecutive. Region G may, for example, comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen TNA (PO) nucleosides, optionally consecutive.

Furthermore, as described herein, other modified nucleosides have been reported as being capable of recruiting RNase H when included within a gap region and may also or alternatively be included in the gap region of a TNA (PO) gapmer. Preferably, in TNA (PO) gapmers comprising at least one, such as one, two or three, TNA (PO) nucleosides or other modified nucleosides in gap region G, gap region G still comprises at least three contiguous DNA nucleosides, such as at least four contiguous DNA nucleosides, such as at least five contiguous DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous DNA nucleosides. In some TNA (PO) gapmers, except for TNA (PO) nucleoside in gap region G, all nucleosides of G are DNA nucleosides.

In some TNA (PO) gapmers, any TNA (PO) nucleosides, particularly any contiguous stretch of two or more TNA or TNA (PO) nucleosides, are located closer to the 5′-end of region G than to the 3′-end of region G. Any contiguous stretch of two or more TNA or TNA (PO) nucleosides in region G may, for example, be located adjacent to the 5′-most nucleoside in region G; typically a DNA nucleoside.

As shown herein, a TNA (PO) gapmer may comprise a stretch of only three or four contiguous DNA nucleosides while still enabling recruitment of RNase H. Without being limited to theory, it is contemplated that a TNA gapmer with a shorter gap region than that of traditional gapmer designs and/or with one or more TNA residues in the gap region may provide for an increased resistance to endonuclease-mediated degradation and/or reduce off-target binding as compared to traditional gapmer designs. Consequently, in some TNA gapmers according to the invention, G comprises at most 10 DNA nucleosides, such as 9, 8, 7, 6, 5, or 4 DNA nucleosides. Optionally, G comprises at most 10 contiguous DNA nucleosides, such as 9, 8, 7, 6, 5, or 4 contiguous DNA nucleosides. Furthermore, as described in more detail further below, when G in a TNA (PO) gapmer comprises 9, 8, 7, 6, 5, or 4 contiguous DNA nucleosides, F′ and F may optionally be of different lengths, e.g., so that F comprises more linked nucleosides than F′.

An antisense gapmer oligonucleotide may, for example, be designed such that, except for the contiguous DNA nucleosides, all other nucleosides are TNA nucleosides, at least one of which linked to at least one adjacent nucleoside by a 2′-PO or 3′-PO internucleoside linkage.

Antisense gapmer oligonucleotides wherein at most about 80%, such as at most about 70%, such as at most about 60%, such as at most about 50%, such as at most about 40%, such as at most about 30%, such as at most about 20%, such as at most about 15%, such as at most about 10%, such as at most about 5%, of the nucleosides of the antisense gapmer oligonucleotide are TNA nucleosides, wherein at least one TNA nucleoside is a 2′-PO linked, 3′-PO linked, or 2′,3′-PO linked, TNA nucleoside, are also contemplated.

Also contemplated are antisense gapmer oligonucleotides wherein at most about 50% of the nucleosides of the antisense gapmer oligonucleotide are TNA nucleosides, wherein at least one TNA nucleoside is a 2′-PO linked, 3′-PO linked, or 2′,3′-PO linked, TNA nucleoside.

Details on the number and placement of at least one TNA (PO) nucleoside in regions F, G and/or F′ have been set out above and can be incorporated into the general gapmer formula 5′-F-G-F′-3′ (I). Specifically contemplated TNA (PO) gapmers include those where

• • (i) region F comprises at least one TNA (PO) nucleoside but regions F′ and G do not;
  • (ii) region F′ comprises at least one TNA (PO) nucleoside but regions F and G do not;
  • (iii) region G comprises at least one TNA (PO) nucleoside but regions F and F′ do not;
  • (iv) region F and F′ each comprises at least one TNA (PO) nucleoside but region G does not;
  • (v) regions F and G each comprises at least one TNA (PO) nucleoside but region F′ does not;
  • (vi) regions G and F′ each comprises at least one TNA (PO) nucleoside but region F does not; and
  • (vii) regions F, G and F′ each comprises at least one TNA (PO) nucleoside.

As an example, in a TNA (PO) gapmer according to item (iv) or (vii), F and F′ may each independently comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA nucleosides, at least one of which is a TNA (PO) nucleoside. Also contemplated are TNA (PO) gapmers according to item (iv) or (vii) where F and F′ each independently comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen contiguous TNA nucleosides at least one of which is a TNA (PO) nucleoside.

Alternatively, in a TNA (PO) gapmer according to any item but (iii), F and F′ may together comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen or at least twenty TNA nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty TNA nucleosides, at least one of which is a TNA (PO) nucleoside.

For example, F and F′ together may together comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty TNA nucleosides, at least one of which is linked to an adjacent TNA nucleoside by a PO internucleoside linkage.

As another example, in a TNA (PO) gapmer according to item (iv) or (vii), F and F′ may each independently comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA (PO) nucleosides. Also contemplated are TNA (PO) gapmers according to item (iv) or (vii) where F and F′ each independently comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen contiguous TNA (PO) nucleosides.

Alternatively, in a TNA (PO) gapmer according to any item but (iii), F and F′ may together comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen or at least twenty TNA (PO) nucleosides, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty TNA (PO) nucleosides.

For example, F and F′ together may together comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty TNA nucleosides which are linked to an adjacent TNA nucleoside by a PO internucleoside linkage.

For example, in a TNA (PO) gapmer according to item (iv), F and F′ may together comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12 TNA (PO) nucleosides.

In some TNA (PO) gapmers, each TNA nucleoside in F and F′ is linked to an adjacent nucleoside by a PO internucleoside linkage. However, also contemplated are TNA (PO) gapmers wherein, except for the 5′-most TNA nucleoside in F and the 3′-most TNA nucleoside in F′, each TNA nucleoside in F and F′ is a TNA (PO) nucleoside.

In some TNA (PO) gapmers, each of region F and F′ may independently comprise or consist of a contiguous sequence of linked sugar-modified nucleosides.

In some TNA (PO) gapmers, at least one of region F and F′ may consist of only one type of sugar modified nucleosides. For example, the sugar modified nucleoside can be a high-affinity nucleoside or a TNA (PO) nucleoside.

In some TNA (PO) gapmers, regions F and F′ may both consist of only one type of sugar modified nucleosides (uniform flanks or uniform gapmer design). For example, the sugar modified nucleoside can be a high-affinity nucleoside or a TNA (PO) nucleoside. In some TNA gapmers, all the nucleosides of regions F and F′ are TNA (PO) nucleosides. In some TNA (PO) gapmers, all the nucleosides of regions F and F′ are sugar-modified nucleosides other than TNA nucleosides.

In some TNA (PO) gapmers, one or both of regions F and F′ may independently comprise two different sugar-modified nucleosides (mixed wing design). One of the two different sugar-modified nucleosides can be a TNA (PO) nucleoside and the other sugar-modified nucleoside can be, for example, a high-affinity nucleoside or a TNA nucleoside linked by a modified internucleoside linkage.

In some TNA (PO) gapmers, all the nucleosides of region F can be TNA (PO) nucleosides. The nucleosides of region F′ may then, for example, comprise or consist of sugar modified nucleosides other than TNA (PO) nucleosides, such as 2′-sugar modified nucleosides, such as high-affinity nucleosides. Optionally, region F′ may comprise two different sugar-modified nucleosides, one of which may be TNA (PO). For example, F′ may comprise 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides, such as three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides. Alternatively, F′ may consist of 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides, such as three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides.

In some TNA (PO) gapmers, all the nucleosides of region F′ can be TNA (PO) nucleosides. The nucleosides of region F may then, for example, comprise or consist of sugar modified nucleosides other than TNA (PO) nucleosides, such as 2′-sugar modified nucleosides, such as high-affinity nucleosides. Optionally, region F may comprise two different sugar-modified nucleosides, one of which may be TNA (PO). For example, F may comprise 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides, such as three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides. Alternatively, F may consist of 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides, such as three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides.

In TNA (PO) gapmers where F, F′ or both F and F′, comprise or consist of one or more sugar-modified nucleosides other than TNA nucleosides, non-limiting examples of sugar-modified nucleosides include those which have modified sugar moiety selected from the group consisting of:

• • 2′-methoxy-ribose (2′-OMe),
  • 2′-O-methoxyethyl-ribose (2′-O-MOE),
  • 5′-methyl-2′-O-methoxyethyl ribose (5′-Me-2′-O-MOE),
  • 2′-O-[2-(methylthio)ethyl]-ribose (2′-O-MTE),
  • 2-(N-methylcarbamoyl)-ethyl]-ribose (2′-O-MCE),
  • 2′-O-[2-(methylamino)-2-oxoethyl]-ribose (2′-O-NMA),
  • 2′-deoxy-2′-fluoro-ribose (as in 2′-deoxy-2′-fluororibo-nucleic acid; 2′-F-RNA),
  • 2′-fluoro-2′-arabinose (as in 2′-fluoro-2′-arabinose nucleic acid; 2′-F-ANA),
  • 2′-O-benzyl-ribose,
  • oxy β-D-locked ribose (as in β-D-LNA),
  • amino β-D-locked ribose (as in amino-β-D-LNA),
  • thio β-D-locked ribose (as in thio-β-D-LNA),
  • oxy β-L-locked ribose (as in β-L-LNA),
  • amino β-L-locked ribose (as in amino-β-L-LNA),
  • thio β-L-locked ribose (as in thio-β-L-LNA),
  • oxy α-L-locked ribose (as in α-L-LNA),
  • amino α-L-locked ribose (as in amino-α-L-LNA),
  • thio α-L-locked ribose (as in thio-α-L-LNA),
  • 2′,4′-constrained 2′-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt),
  • tricyclo-deoxyribose (as in tricyclo-deoxyribose DNA; TcDNA),
  • 3′-deoxy-ribose (as in 3′-deoxy-ribose DNA; 3′-DNA),
  • unlocked ribose (as in unlocked nucleic acid; UNA),
  • glycol (as in glycol nucleic acid; GNA),
  • hexitol (as in hexitol nucleic acid; HNA),
  • 3′-fluoro hexitol (as in 3′-fluoro hexitol nucleic acid; FHNA),
  • 3′-arabino-fluoro hexitol (as in 3′-arabino-gluoro hexitol nucleic acid; Ara-FHNA),
  • cyclohexene (as in cyclohexene nucleic acid; CeNA),
  • fluoro-cyclohexenenyl (as in 2′-fluoro-cyclohexenyl nucleic acid; F-CeNA),
  • serinol (as in serinol nucleic acid; SNA),
  • 2′-O,4′-C-ethylene bridged ribose (as in 2′-O,4′-C-ethylene linked nucleic acid; ENA),
  • acyclic (L)-threoninol (as in acyclic (L)-threoninol nucleic acid; aTNA),
  • 2′,4′-constrained 2′-O-methoxyethyl ribose (as in cMOE), and
  • 7′,5′-alpha-bicyclo sugar unit (as in 7′,5′-alpha-bicyclo DNA; bcDNA).

Particularly contemplated are TNA (PO) gapmers wherein the sugar-modified nucleosides of F, F′ or both F and F′, comprise or consist of one or more 2′-sugar modified nucleosides, such as high-affinity 2′-sugar-modified nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more LNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be LNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 LNA nucleosides, such as 3, 4 or 5 LNA nucleosides. Suitable LNA nucleosides include those which have a modified sugar moiety selected from oxy, amino or thio β-D-locked ribose (B-D-LNA) or from oxy, amino or thio β-L-locked ribose (B-L-LNA) or from oxy, amino or thio α-L-locked ribose (α-L-LNA), such as β-D-oxy-LNA, e.g., 6′-methyl-β-D-oxy LNA such as(S)-6′-methyl-β-D-oxy-LNA (ScET), and ENA as well as the LNA nucleosides disclosed in Scheme 2. A particularly contemplated LNA nucleoside is β-D-oxy-LNA.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more MOE nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be MOE nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 MOE nucleosides, such as 3, 4 or 5 MOE nucleosides. Suitable MOE nucleosides include 2′-O-MOE, 5′-Me-2′-O-MOE and 2′,4′-constrained 2′-O-methoxyethyl (cMOE). A particularly contemplated MOE nucleoside is 2′-O-MOE.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more 2′-OMe nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be 2′-OMe nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2′-OMe nucleosides, such as 3, 4 or 5 2′-OMe nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more 2′-O-MTE nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be 2′-O-MTE nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2′-O-MTE nucleosides, such as 3, 4 or 5 2′-O-MTE nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more 2′-O-MCE nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be 2′-O-MCE nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2′-O-MCE nucleosides, such as 3, 4 or 5 2′-O-MCE nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more 2′-O-NMA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be 2′-O-NMA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more

TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2′-O-NMA nucleosides, such as 3, 4 or 5 2′-O-NMA nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more 2′-deoxy-2′-fluoro-ribose nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be 2′-deoxy-2′-fluoro-ribose nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2′-deoxy-2′-fluoro-ribose nucleosides, such as 3, 4 or 5 2′-deoxy-2′-fluoro-ribose nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more 2′-fluoro-2′-arabinose nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be 2′-fluoro-2′-arabinose nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2′-fluoro-2′-arabinose nucleosides, such as 3, 4 or 5 2′-fluoro-2′-arabinose nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more 2′-O-benzyl-ribose nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be 2′-O-benzyl-ribose nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 2′-O-benzyl-ribose nucleosides, such as 3, 4 or 5 2′-O-benzyl-ribose nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more cEt nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be cEt nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 cEt nucleosides, such as 3, 4 or 5 cEt nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more TcDNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be TcDNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 TcDNA nucleosides, such as 3, 4 or 5 TcDNA nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more 3′-DNA nucleosides. For example, except for any at least one

TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be 3′-DNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 3′-DNA nucleosides, such as 3, 4 or 5 3′-DNA nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more UNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be UNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 UNA nucleosides, such as 3, 4 or 5 UNA nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more GNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be GNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 GNA nucleosides, such as 3, 4 or 5 GNA nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more HNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be HNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 HNA nucleosides, such as 3, 4 or 5 HNA nucleosides. Suitable HNA nucleosides include HNA, FHNA, and Ara-FHNA.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more CeNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be CeNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 CeNA nucleosides, such as 3, 4 or 5 CeNA nucleosides. Suitable CeNA nucleosides include CeNA and F-CeNA.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more SNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be SNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 SNA nucleosides, such as 3, 4 or 5 SNA nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more ENA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be ENA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 ENA nucleosides, such as 3, 4 or 5 ENA nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more SNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be SNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 SNA nucleosides, such as 3, 4 or 5 SNA nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more aTNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be aTNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 aTNA nucleosides, such as 3, 4 or 5 aTNA nucleosides.

In some TNA (PO) gapmers, the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more bcDNA nucleosides. For example, except for any at least one TNA (PO) nucleoside, 1, 2, 3, 4, 5, 6, 7, 8 or all nucleosides in F′, F, or both F and F′, may be bcDNA nucleosides. In a TNA (PO) gapmer where only region G comprises one or more TNA (PO) nucleosides, regions F and F′ may, for example, each comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8 bcDNA nucleosides, such as 3, 4 or 5 bcDNA nucleosides.

For any TNA (PO) gapmer described herein, particularly TNA (PO) gapmers wherein the sugar-modified nucleosides of F, F′ or both F and F′, comprise or consist of one or more 2′-sugar modified nucleosides, such as high-affinity 2′-sugar-modified nucleosides, it is contemplated that at least one internucleoside linkage may be a PS₂ internucleoside linkage. For example, in some TNA (PO) gapmers, (i) the 5′-most nucleoside of F is linked to the adjacent nucleoside in F by a PS₂ internucleoside linkage, (ii) the 3′-most nucleoside of F′ is linked to the adjacent nucleoside in F′ by a PS₂ internucleoside linkage, or (iii) both (i) and (ii) apply. Optionally, other nucleosides than the at least one TNA (PO) nucleoside(s) in the TNA (PO) gapmer can be linked to each other by PS internucleoside linkages.

Particularly contemplated are TNA (PO) gapmers wherein the contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I) has a length of from 12 to 32 nucleosides, such as from 12 to 28 nucleosides, such as from 12 to 26 nucleosides, such as from 14 to 26 nucleosides, such as from 14 to 24 nucleosides, such as from 14 to 22 nucleosides, such as from 16 to 22 nucleosides, such as from 16 to 20 nucleosides. However, any suitable length can be used for the F-G-F′ design, including, but limited to, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32 linked nucleosides.

By way of example, a TNA (PO) gapmer according to the present invention can be represented by one or more of the following formulae for regions F-G-F′ (Formula I), with the proviso that the overall length of regions F-G-F′ is at least 12, such as at least 14 nucleotides in length:

F 1 ⁢ ‐ ⁢ 15 ⁢ ‐ ⁢ G 3 ⁢ ‐ ⁢ 18 ⁢ ‐ ⁢ F 1 ⁢ ‐ ⁢ 15 ′ ( IV ) , such ⁢ as ⁢ F 1 ⁢ ‐ ⁢ 15 ⁢ ‐ ⁢ G 3 ⁢ ‐ ⁢ 18 ⁢ ‐ ⁢ F 1 ⁢ ‐ ⁢ 12 ′ ( IVa ) , 
 or ⁢ F 1 ⁢ ‐ ⁢ 12 ⁢ ‐ ⁢ G 3 ⁢ ‐ ⁢ 18 ⁢ ‐ ⁢ F 1 ⁢ ‐ ⁢ 15 ′ ( IVb ) ; F 1 ⁢ ‐ ⁢ 12 ⁢ ‐ ⁢ G 3 ⁢ ‐ ⁢ 18 ⁢ ‐ ⁢ F 1 ⁢ ‐ ⁢ 12 ′ ( V ) , such ⁢ as ⁢ F 1 - 12 ⁢ ‐ ⁢ G 3 ⁢ ‐ ⁢ 18 ⁢ ‐ ⁢ F 1 ⁢ ‐ ⁢ 9 ′ ( Va ) , 
 or ⁢ F 1 - 9 ⁢ ‐ ⁢ G 3 ⁢ ‐ ⁢ 18 ⁢ ‐ ⁢ F 1 ⁢ ‐ ⁢ 12 ′ ( Vb ) ; F 1 ⁢ ‐ ⁢ 12 ⁢ ‐ ⁢ G 4 ⁢ ‐ ⁢ 16 ⁢ ‐ ⁢ F 1 ⁢ ‐ ⁢ 12 ′ ( VI ) , such ⁢ as ⁢ F 1 ⁢ ‐ ⁢ 12 ⁢ ‐ ⁢ G 4 ⁢ ‐ ⁢ 16 ⁢ ‐ ⁢ F 1 ⁢ ‐ ⁢ 9 ′ ( VIa ) , 
 or ⁢ F 1 ⁢ ‐ ⁢ 9 ⁢ ‐ ⁢ G 4 ⁢ ‐ ⁢ 16 ⁢ ‐ ⁢ F 1 ⁢ ‐ ⁢ 12 ′ ( VIb ) ; and F 3 ⁢ ‐ ⁢ 12 ⁢ ‐ ⁢ G 4 ⁢ ‐ ⁢ 10 ⁢ ‐ ⁢ F 3 ⁢ ‐ ⁢ 12 ′ ( VII ) , such ⁢ as ⁢ F 3 ⁢ ‐ ⁢ 12 ⁢ ‐ ⁢ G 4 ⁢ ‐ ⁢ 10 ⁢ ‐ ⁢ F 3 ⁢ ‐ ⁢ 9 ′ ( VIIa ) , 
 or ⁢ F 3 ⁢ ‐ ⁢ 9 ⁢ ‐ ⁢ G 4 ⁢ ‐ ⁢ 10 ⁢ ‐ ⁢ F 3 ⁢ ‐ ⁢ 12 ′ ( VIIb ) .

Each of region F, G and F′ as described herein can be incorporated into any of the F-G-F′ formulae.

In some TNA (PO) gapmers, the contiguous nucleotide sequence, e.g., of formula IVa, has a length of at least 16 nucleosides and

• • (a) the 5′-most nucleosides in F and the nucleosides in F′ are independently 3, 4 or 5 high-affinity sugar-modified nucleosides,
  • (b) the remaining nucleosides in F are TNA (PO) nucleosides, and
  • (c) all nucleosides in G are DNA nucleosides.

For example, G may comprise at most 10 contiguous DNA nucleosides, such as 9, 8, 7, 6, 5, or 4 contiguous DNA nucleosides, such as 4, 5 or 6 contiguous DNA nucleosides. F may, for example, comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 contiguous TNA (PO) nucleosides.

In some TNA (PO) gapmers, the contiguous nucleotide sequence, e.g. of formula IV, has a length of at least 16 nucleosides and

• • (a) F and F′ each independently consists of 3, 4, or 5 nucleosides, wherein at least one of the nucleosides in F and/or F′ is a TNA (PO) nucleoside and any remaining nucleosides are high-affinity sugar-modified nucleosides, and
  • (b) all nucleosides in G are DNA nucleosides.

For example, all nucleosides in either F or F′ may be TNA (PO) nucleosides. Alternatively, all nucleosides in F and F′ may be TNA (PO) nucleosides. Alternatively, except for the 5′-most TNA nucleoside in F and the 3′-most TNA nucleoside in F′, each TNA nucleoside in F and F′ may be linked to an adjacent nucleoside, particularly to any adjacent TNA nucleoside, by a PO internucleoside linkage.

In some TNA (PO) gapmers, the contiguous nucleotide sequence, e.g. of formula IV, has a length of at least 16 nucleosides and

• • (a) F and F′ each independently comprises or consists of 3, 4, or 5 linked high-affinity sugar-modified nucleosides and does not comprise any TNA nucleoside, and
  • (b) the second, third, fourth or fifth 5′-most nucleoside in G is a TNA (PO) nucleoside and the remaining nucleosides in G are DNA nucleosides.

The TNA (PO) nucleoside may, for example, be located closer to the 5′-end of region G than to the 3′-end of G.

Particularly contemplated designs of TNA (PO) gapmers include also those referred to as Design A and Design B in Example 1:

• • TTTTTddddddddddTTTTT (Design A), where T is TNA and d is DNA, and with the backbone (internucleoside linkage) pattern sooosssssssssssooOs, where S corresponds to phosphorothioate (PS), and O to phosphodiester (PO).
  • MMMMMTTTTTTddddMMMMM (Design B), where M is MOE (e.g., 2′-O-MOE), T is TNA and d is DNA, and with the backbone (internucleoside linkage) pattern SSSSOOOOOOSSSSSSSSS, where S corresponds to phosphorothioate (PS), and O to phosphodiester (PO).

Also particularly contemplated are the following designs:

• • TTTTTTTTTTdddddMMMMM (Design C), where M is 2′-O-MOE, T is TNA and d is DNA, and with the backbone (internucleoside linkage) pattern 000000000SSSSSSSSSS, where S corresponds to phosphorothioate (PS), and O to phosphodiester (PO).
  • MMMMMddddddddddMMMMM, wherein one, two, three, four, five six or seven d's are replaced by T's (Design D), where M is 2′-O-MOE, T is TNA and d is DNA, and with the backbone (internucleoside linkage) pattern being SSSSSSSSSSSSSSSSSSS, except for O replacing S as the 2′- and 3′-internucleoside linkage for each T, where S corresponds to phosphorothioate (PS), and O to phosphodiester (PO).

Further particularly contemplated are the following designs:

• • TTTTTddddddddddTTTTT (Design A′), where T is TNA and d is DNA, and with the backbone (internucleoside linkage) pattern POOOSSSSSSSSSSSOOOP, where P corresponds to PS₂, where S corresponds to phosphorothioate (PS), and O to phosphodiester (PO).
  • MMMMMTTTTTTddddMMMMM (Design B′), where M is MOE (e.g., 2′-O-MOE), T is TNA and d is DNA, and with the backbone (internucleoside linkage) pattern PSSSOOOOOOSSSSSSSSP, where P corresponds to PS₂, S corresponds to phosphorothioate (PS), and O to phosphodiester (PO).
  • TTTTTTTTTTdddddMMMMM (Design C′), where M is 2′-O-MOE, T is TNA and d is DNA, and with the backbone (internucleoside linkage) pattern 000000000SSSSSSSSSP, where P corresponds to PS₂, S corresponds to phosphorothioate (PS), and O to phosphodiester (PO).
  • MMMMMddddddddddMMMMM, wherein one, two, three, four, five six or seven d's are replaced by T's (Design D′), where M is 2′-O-MOE, T is TNA and d is DNA, and with the backbone (internucleoside linkage) pattern being PSSSSSSSSSSSSSSSSSP, except for O replacing S as any 2′- and 3′-internucleoside linkage for each T, where S corresponds to phosphorothioate (PS), O to phosphodiester (PO), and P to phosphorodithioate (PS₂).

An antisense gapmer oligonucleotide comprising a TNA (PO) gapmer, i.e., contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I), may comprise additional linked nucleosides, e.g., from 1 to 100, 1 to 40, 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 linked nucleosides, at the 3′- and/or 5′-end of the TNA (PO) gapmer. The additional linked nucleosides may, for example, facilitate delivery of the antisense gapmer oligonucleotide to the intended site or target a second molecule. The antisense gapmer oligonucleotide can also be, or can be part of, a longer nucleic acid construct. However, it is also contemplated that the antisense gapmer oligonucleotide may consist of the TNA (PO) gapmer.

Particularly contemplated for the present invention are TNA (PO) gapmers and antisense gapmer oligonucleotides, which are single-stranded antisense oligonucleotides. In a preparation of TNA (PO) gapmers or antisense gapmer oligonucleotides according to the invention, the TNA (PO) gapmers or antisense gapmer oligonucleotides are essentially single stranded, such that the majority of the TNA (PO) gapmer molecules or antisense gapmer oligonucleotide molecules are in single-stranded form.

Method of Manufacture

Provided are also methods for manufacturing the oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313).

The synthesis of TNA monomers and their incorporation into oligonucleotides are disclosed in, e.g., Zhang and Chaput, “Synthesis of Threose Nucleic Acid (TNA) Phosphoramidite Monomers and Oligonucleotide Polymers, Current Protocols in Nucleic Acid Chemistry, 4.51.1-4.51.26, 2012”, WO 2012/078536, WO 2012/118911 and WO 2013/179292 A1.

Particularly provided is a method of preparing a modified version of a parent antisense gapmer oligonucleotide, wherein the parent antisense gapmer comprises a contiguous nucleotide sequence of formula 5′ F-G-F′ 3′ (I) which is capable of recruiting RNase H, wherein G is a gap region of 5 to 18 linked DNA nucleosides and each of F and F′ is a flanking region of up to 8 linked nucleosides which independently comprises or consists of 1 to 8 sugar-modified nucleosides other than TNA nucleosides, and wherein, in the modified version, at least one nucleoside in F, F′, and/or G of the parent antisense gapmer oligonucleotide has been replaced with a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage,

• • the method comprising the step of manufacturing the modified antisense gapmer oligonucleotide by reacting nucleotide units to form covalently linked contiguous nucleotide units comprised in the oligonucleotide, wherein at least one of the nucleotide units comprises a TNA nucleoside which is linked to an adjacent nucleoside by a PO internucleoside bond, and,
  • optionally purifying or isolating the modified antisense gapmer oligonucleotide.

In one embodiment, the modified antisense gapmer oligonucleotide has a reduced toxicity, optionally hepatotoxicity, as compared to the parent antisense gapmer oligonucleotide. In one embodiment, the modified antisense gapmer oligonucleotide is less toxic to HepG2 cells than the parent antisense gapmer oligonucleotide, optionally as determined by a Caspase 3/7 assay. In one embodiment, the modified antisense gapmer oligonucleotide has an increased exonuclease resistance as compared to the parent antisense gapmer oligonucleotide. In one embodiment, the modified antisense gapmer oligonucleotide has an increased endonuclease resistance as compared to the parent antisense gapmer oligonucleotide.

Optionally, the parent antisense gapmer oligonucleotide can be an LNA gapmer or MOE gapmer, such as an LNA gapmer or MOE gapmer wherein all internucleoside linkages are phosphorothioate linkages. Furthermore, the nucleotide units employed in the manufacturing step are advantageously nucleoside phosphoramidites.

The modified antisense gapmer oligonucleotide may comprise the features of any TNA (PO) gapmer described herein, e.g., as to regions F, G and F′ and the F-G-F′ design.

Also provided by the present invention is an antisense gapmer oligonucleotide obtained or obtainable by the method.

The method may further comprise reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide.

In a further aspect, a method is provided for manufacturing a composition, comprising mixing the oligonucleotide or conjugated oligonucleotide with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

Pharmaceutical Composition

In a further aspect, the invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.

In a further aspect, the invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments the pharmaceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments, the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50-300 μM solution.

The oligonucleotides or oligonucleotide conjugates according to the present invention may exist in the form of their pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin, Organic Process Research & Development 2000, 4, 427-435 or in Ansel, In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example, the pharmaceutically acceptable salt of the compounds provided herein may be a sodium salt.

Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990). WO 2007/031091 provides further suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (hereby incorporated by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in WO2007/031091.

Oligonucleotides or oligonucleotide conjugates of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. In particular with respect to oligonucleotide conjugates the conjugate moiety is cleaved of the oligonucleotide once the prodrug is delivered to the site of action, e.g., the target cell.

Applications

The oligonucleotides or oligonucleotide conjugates described herein may be utilized as research reagents or as diagnostic, therapeutic and prophylactic agents.

In research, the oligonucleotides or oligonucleotide conjugates may be used to specifically modulate the expression of a target nucleic acid in cells (e.g., in in vitro cell cultures) and experimental animals, thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention. Typically, the target modulation is achieved by degrading or inhibiting the mRNA producing the protein, thereby preventing protein formation or by degrading or inhibiting a modulator of the gene or mRNA producing the protein.

If employing the oligonucleotide or oligonucleotide conjugate in research or diagnostics, the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

Also provided are in vivo or in vitro methods for modulating the expression of a target gene in a target cell, which comprises a target nucleic acid, the method comprising administering an oligonucleotide or oligonucleotide conjugate of the invention in an effective amount to said cell.

In some embodiments, the target cell is a mammalian cell, in particular a human cell. The target cell may be an in vitro cell culture or an in vivo cell forming part of a tissue in a mammal, such as a human.

Also provided are diagnostic applications in which the oligonucleotides or oligonucleotide conjugates may be used to detect and quantitate the expression of a target gene in cells and tissues by northern blotting, in-situ hybridisation or similar techniques.

Also provided is an oligonucleotide, an oligonucleotide conjugate, or a pharmaceutical composition as described herein for use as a medicament.

The disease or disorder in which the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition is used is typically associated with expression of the target gene. Preferably, the disease or disorder is one, which may be treated by modulating the expression of the target gene.

The oligonucleotide, an oligonucleotide conjugate, or a pharmaceutical composition may, for example, be employed for treatment or prophylaxis against diseases or disorders caused by abnormal levels and/or activity of the target gene or an expression product from the target gene, e.g., an RNA or protein. The disease or disorder may also or alternatively be associated with a mutation in the target gene. Therefore, in some embodiments, the target nucleic acid is a mutated form of the target gene. Non-limiting examples of target genes include genes associated with one or more cancers, infectious diseases, neurological diseases or disorders, ophthalmic diseases or disorders, or cardiovascular diseases or disorders.

The oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.

Also provided are therapeutic applications wherein the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition is for use in treating or preventing a disease or disorder in an animal or a human suffering from or suspected of having the disease or disorder. Typically, the disease or disorder is one, which can be treated by modulating the expression of the target gene.

Also provided are uses of the oligonucleotide or oligonucleotide conjugate in the manufacture of a medicament for treating or preventing a disease or disorder in an animal or a human suffering from or suspected of having the disease or disorder. Typically, the disease or disorder is one, which can be treated by modulating the expression of the target gene.

Also provided are methods for treating or preventing a disease or disorder, comprising administering a therapeutically or prophylactically effective amount of the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition to a subject suffering from or susceptible to the disease or disorder.

Administration

In some embodiments, the oligonucleotides or pharmaceutical compositions of the present invention may be administered orally. In further embodiments, the oligonucleotides or pharmaceutical compositions of the present invention may be administered topically or enterally or parenterally (such as, intravenously, subcutaneously, intra-muscularly, intracerebrally, intracerebroventricularly or intrathecally).

In a preferred embodiment the oligonucleotide or pharmaceutical compositions of the present invention are administered by a parenteral route, such as, for example, by intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intrathecal or intracranial administration, e.g., intracerebral or intraventricular administration, or intravitreal administration. In one embodiment, the active oligonucleotide or oligonucleotide conjugate is administered intravenously. In another embodiment, the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.

In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is administered at a dose of 0.1-15 mg/kg, such as from 0.2-10 mg/kg, such as from 0.25-5 mg/kg. The administration can be once a week, every 2nd week, every third week or even once a month.

Combination Therapies

In some embodiments the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent. The therapeutic agent can for example be the standard of care for the diseases or disorders to be treated with the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention.

EMBODIMENTS

The following numbered embodiments are particularly contemplated.

1. An antisense gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I) which is capable of recruiting ribonuclease (RNase) H, wherein

• • G is a gap region of up to 18 linked nucleosides which comprises at least 3 contiguous DNA nucleosides,
  • each of F and F′ is a flanking region of up to 15 linked nucleosides which independently comprises or consists of 1 to 15 sugar-modified nucleosides,
  • at least one of F, F′ and G comprises a sugar-modified nucleoside which is an α-L-threofuranosyl (TNA) nucleoside and which is linked to an adjacent nucleoside by an internucleoside linkage different than a phosphorothioate (PS) internucleoside linkage.

2. The antisense gapmer oligonucleotide according to embodiment 1, wherein the TNA nucleoside is linked to an adjacent nucleoside by a phosphodiester (PO) internucleoside linkage.

3. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the TNA nucleoside is linked to an adjacent nucleoside by a 2′-PO internucleoside linkage or a 3′-PO internucleoside linkage.

4. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the TNA nucleoside is linked to any adjacent nucleoside by a 2′-PO or 3′-PO internucleoside linkage.

5. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the adjacent nucleoside is a sugar-modified nucleoside or a DNA nucleoside.

6. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the TNA nucleoside is linked to a first and a second adjacent nucleoside by

• • (a) a 2′-PO internucleoside linkage and a 3′-modified internucleoside linkage, respectively;
  • (b) a 2′-modified internucleoside linkage and a 3′-PO internucleoside linkage, respectively; or
  • (c) a 2′-PO internucleoside linkage and a 3′-PO internucleoside linkage, respectively,
  • optionally wherein the 2′-modified internucleoside linkage or 3′-modified internucleoside linkage is a phosphorothioate (PS) internucleoside linkage or a phosphorodithioate (PS₂) internucleoside linkage.

7. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F comprises at least one TNA nucleoside.

8. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

9. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA nucleosides linked to an adjacent nucleoside by a PO internucleoside linkage.

10. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA nucleosides linked by PO internucleoside linkages.

11. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F comprises or consists of two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA nucleosides linked by PO internucleoside linkages.

12. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the 3′-most nucleoside in F is a TNA nucleoside.

13. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 3′-most nucleosides in F are TNA nucleosides.

14. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen 3′-most nucleosides in F are TNA nucleosides.

15. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the 5′-most nucleoside in F is a TNA nucleoside.

16. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 5′-most nucleosides in F are TNA nucleosides.

17. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen 5′-most nucleosides in F are TNA nucleosides.

18. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein all sugar-modified nucleosides of F are TNA nucleosides.

19. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein all nucleosides of F are TNA nucleosides.

20. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein

• • (a) each TNA nucleoside in F is linked to an adjacent nucleoside by a PO internucleoside linkage; or
  • (b) except for the 5′-most TNA nucleoside in F, each TNA nucleoside in F is linked to an adjacent nucleoside by a PO internucleoside linkage.

21. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 20, wherein F′ does not comprise any TNA nucleoside.

22. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 20, wherein F′ comprises at least one TNA nucleoside.

23. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F′ comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

24. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F′ comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve TNA nucleosides linked to an adjacent nucleoside by a PO internucleoside linkage.

25. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F′ comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA nucleosides linked by PO internucleoside linkages.

26. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F′ comprises or consists of two, three, four, five, six, seven, eight, nine, ten, eleven or twelve adjacent TNA nucleosides linked by PO internucleoside linkages.

27. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the 5′-most nucleoside in F′ is a TNA nucleoside.

28. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 5′-most nucleosides in F′ are TNA nucleosides.

29. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen 5′-most nucleosides in F′ are TNA nucleosides.

30. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the 3′-most nucleoside in F′ is a TNA nucleoside.

31. The antisense oligonucleotide according to any one of the preceding embodiments, wherein at least the two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 3′-most nucleosides in F′ are TNA nucleosides.

32. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen 3′-most nucleosides in F′ are TNA nucleosides.

33. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein all sugar-modified nucleosides of F′ are TNA nucleosides.

34. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein all nucleosides of F′ are TNA nucleosides.

35. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein

• • (a) each TNA nucleoside in F′ is linked to an adjacent nucleoside by a PO internucleoside linkage; or
  • (b) except for the 3′-most TNA nucleoside in F′, each TNA nucleoside in F′ is linked to an adjacent nucleoside by a PO internucleoside linkage.

36. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 6 and 21 to 35, wherein F does not comprise any TNA nucleoside.

37. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F and F′ each independently comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen TNA nucleosides linked to an adjacent nucleoside by a PO internucleoside linkage.

38. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F and F′ together comprise at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty TNA nucleosides linked to an adjacent nucleoside by a PO internucleoside linkage.

39. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F and F′ each independently comprises or consists of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen adjacent TNA nucleosides linked by PO internucleoside linkages.

40. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F and F′ together comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty TNA nucleosides which are linked to an adjacent TNA nucleoside by a PO internucleoside linkage.

41. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein

• • (a) each TNA nucleoside in F and F′ is linked to an adjacent nucleoside by a PO internucleoside linkage, or
  • (b) except for the 5′-most TNA nucleoside in F and the 3′-most TNA nucleoside in F′, each TNA nucleoside in F and F′ is linked to an adjacent nucleoside by a PO internucleoside linkage.

42. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein F, F′, or both F and F′, further comprise 1 to 8 sugar-modified nucleosides other than TNA (PO) nucleosides, such as three, four or five sugar-modified nucleosides other than TNA (PO) nucleosides.

43. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the sugar-modified nucleosides of F, F′ or both F and F′, comprise or consist of at least one sugar-modified nucleoside comprising a modified sugar-moiety selected from the group consisting of:

• • α-L-threofuranosyl (as in threose nucleic acid; TNA),
  • 2′-methoxy-ribose (2′-OMe),
  • 2′-O-methoxyethyl-ribose (2′-O-MOE),
  • 5′-methyl-2′-O-methoxyethyl ribose (5′-Me-2′-O-MOE),
  • 2′-O-[2-(methylthio)ethyl]-ribose (2′-O-MTE),
  • 2-(N-methylcarbamoyl)-ethyl]-ribose (2′-O-MCE),
  • 2′-O-[2-(methylamino)-2-oxoethyl]-ribose (2′-O-NMA),
  • 2′-deoxy-2′-fluoro-ribose (as in 2′-deoxy-2′-fluororibo-nucleic acid; 2′-F-RNA),
  • 2′-fluoro-2′-arabinose (as in 2′-fluoro-2′-arabinose nucleic acid; 2′-F-ANA),
  • 2′-O-benzyl-ribose,
  • oxy β-D-locked ribose (as in β-D-LNA),
  • amino β-D-locked ribose (as in amino-β-D-LNA),
  • thio β-D-locked ribose (as in thio-β-D-LNA),
  • oxy β-L-locked ribose (as in β-L-LNA),
  • amino β-L-locked ribose (as in amino-β-L-LNA),
  • thio β-L-locked ribose (as in thio-β-L-LNA),
  • oxy α-L-locked ribose (as in α-L-LNA),
  • amino α-L-locked ribose (as in amino-α-L-LNA),
  • thio α-L-locked ribose (as in thio-α-L-LNA),
  • 2′,4′-constrained 2′-O-ethyl ribose (as in constrained ethyl locked nucleic acid; cEt),
  • tricyclo-deoxyribose (as in tricyclo-deoxyribose DNA; TcDNA),
  • 3′-deoxy-ribose (as in 3′-deoxy-ribose DNA; 3′-DNA),
  • unlocked ribose (as in unlocked nucleic acid; UNA),
  • glycol (as in glycol nucleic acid; GNA),
  • hexitol (as in hexitol nucleic acid; HNA),
  • 3′-fluoro hexitol (as in 3′-fluoro hexitol nucleic acid; FHNA),
  • 3′-arabino-fluoro hexitol (as in 3′-arabino-gluoro hexitol nucleic acid; Ara-FHNA),
  • cyclohexene (as in cyclohexene nucleic acid; CeNA),
  • fluoro-cyclohexenenyl (as in 2′-fluoro-cyclohexenyl nucleic acid; F-CeNA),
  • serinol (as in serinol nucleic acid; SNA),
  • 2′-O,4′-C-ethylene bridged ribose (as in 2′-O,4′-C-ethylene linked nucleic acid; ENA)
  • acyclic (L)-threoninol (as in acyclic (L)-threoninol nucleic acid; aTNA)
  • 2′,4′-constrained 2′-O-methoxyethyl ribose (as in cMOE)
  • 7′,5′-alpha-bicyclo sugar unit (as in 7′,5′-alpha-bicyclo DNA; bcDNA).

44. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the sugar-modified nucleosides of F, F′ or both F and F′, comprise or consist of one or more 2′-sugar modified nucleosides, such as high-affinity 2′-sugar-modified nucleosides.

45. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the sugar-modified nucleosides of F, F′ or both F and F′, comprise one or more LNA nucleosides.

46. The antisense gapmer oligonucleotide according to embodiment 45, wherein, except for any at least one TNA nucleoside, all nucleosides in F′ and F are LNA nucleosides.

47. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the sugar-modified nucleosides of F, F′, or both F and F′, comprise one or more 2′-O-methoxyethyl-RNA (2′-O-MOE) nucleosides.

48. The antisense gapmer oligonucleotide according to embodiment 47, wherein, except for any at least one TNA nucleoside, all nucleosides in F′ and F are 2′-O-MOE nucleosides.

49. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein G does not comprise any TNA nucleoside.

50. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 48, wherein G comprises at least one TNA nucleoside, such as at least one, two, or three, TNA nucleosides.

51. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein G comprises at least one TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

52. The antisense gapmer oligonucleotide according to embodiment 51, wherein the second, third, fourth, fifth, sixth, seventh-most, or eight-most 5′ nucleoside in G is a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

53. The antisense gapmer oligonucleotide according to embodiment 51 or 52, wherein the second, third, fourth, fifth, sixth, seventh-most, or eight-most 3′ nucleoside in G is a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage.

54. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein G comprises at least two, such as two or three, adjacent TNA nucleosides linked by PO internucleoside linkages.

55. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein at least the 5′-most and 3′-most nucleosides in G are DNA nucleosides.

56. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein, except for any at least one TNA (PO) nucleoside, all nucleosides of G are DNA nucleosides.

57. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the gap region G comprises at least four DNA nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous DNA nucleosides.

58. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein G comprises at most 10 DNA nucleosides, such as 9, 8, 7, 6, 5, or 4 DNA nucleosides.

59. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein at most about 80%, such as at most about 70%, such as at most about 60%, such as at most about 50%, such as at most about 40%, such as at most about 30%, such as at most about 20%, such as at most about 15%, such as at most about 10%, such as at most about 5%, of the nucleosides of the antisense gapmer oligonucleotide are TNA nucleosides.

60. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein at most about 50% of the nucleosides of the antisense gapmer oligonucleotide are TNA nucleosides, wherein at least one TNA nucleoside is a 2′-PO linked, 3′-PO linked, or 2′,3′-PO linked, TNA nucleoside.

61. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, which comprises at least one modified internucleoside linkage.

62. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, which comprises a nuclease resistant modified internucleoside linkage.

63. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein, except for any PO internucleoside linkage between a TNA nucleoside and an adjacent nucleoside, all internucleoside linkages are phosphorothioate (PS) internucleoside linkages, phosphorodithioate (PS₂) internucleoside linkages, or a combination thereof.

64. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein

• • (a) the 5′-most nucleoside of F is linked to the adjacent nucleoside in F by a PS₂ internucleoside linkage;
  • (b) the 3′-most nucleoside of F′ is linked to the adjacent nucleoside in F′ by a PS₂ internucleoside linkage, or
  • (c) both (a) and (b).

65. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, wherein the contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I) has a length of from 12 to 32 nucleosides, such as from 12 to 28 nucleosides, such as from 12 to 26 nucleosides, such as from 14 to 26 nucleosides, such as from 14 to 24 nucleosides, such as from 14 to 22 nucleosides, such as from 16 to 22 nucleosides, such as from 16 to 20 nucleosides.

66. The antisense gapmer oligonucleotide according to embodiment 65, wherein the contiguous nucleotide sequence is of the formula

• • F_1-15-G_3-18-F′_1-15 (IV), such as F_1-15-G_3-18-F′_1-12 (IVa) or F_1-12-G_3-18-F′_1-15 (IVb);
  • F_1-12-G_3-18-F′_1-12 (V), such as F_1-12-G_3-18-F′_1-9 (Va), or F_1-9-G_3-18-F′_1-12 (Vb);
  • F_1-12-G_4-16-F′_1-12 (VI), such as F_1-12-G_4-16-F′_1-9 (VIa), or F_1-9-G_4-16-F′_1-12 (VIb); or
  • F_3-12-G_4-10-F′_3-12 (VII), such as F_3-12-G_4-10-F′_3-9 (VIIa) or F_3-9-G_4-10-F′_3-12 (VIIb), wherein the numeric ranges represent the number of linked nucleosides in F, G and F′, respectively.

67. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, which can reduce the expression level of a target nucleic acid by at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, as compared to the normal expression level of the target, optionally when determined in a target cell expressing the target nucleic acid and incubated with the antisense gapmer oligonucleotide at a concentration of about 25 μM for about 3 days.

68. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, which antisense gapmer oligonucleotide according to any one of the preceding embodiments, which has an IC50 of no more than about 20 μM, such as no more than about 10 μM, such as no more than about 5 μM, for reducing the expression level of a target nucleic acid, optionally when determined in a target cell expressing the target nucleic acid and incubated with the antisense gapmer oligonucleotide at a concentration of about 25 μM for about 3 days.

69. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, which using the Caspase 3/7 assay described in Example 4, the percentage assay window (% AW) is preferably at most about 60%, such as at most about 40%, such as at most about 20%, such as at most about 10%.

70. The antisense gapmer oligonucleotide according to any one of the preceding embodiments, which has, in the form of a duplex of the antisense gapmer oligonucleotide with an RNA target sequence, a melting temperature (Tm) of at least about 50° C., such as at least about 52° C., such as at least about 54° C., such as at least about 56° C., such as at least about 58° C., such as at least about 60° C.

71. The antisense gapmer oligonucleotide according to any one of embodiments 1 to 66, which is characterized by a combination of embodiments 67 and 68; embodiments 67 and 69; embodiments 67 and 70; embodiments 68 and 69; embodiments 68 and 70; embodiments 67, 68 and 69; embodiments 67, 68 and 70; embodiments 67, 69 and 70, embodiments 68, 69, and 70; or embodiments 67 to 70.

72. The antisense gapmer oligonucleotide according to any one of embodiments 67 to 71, wherein the target nucleic acid is an RNA target sequence which has a nucleobase sequence complementary to the contiguous nucleotide sequence of formula I.

73. An antisense gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I) which is capable of recruiting RNase H, wherein the contiguous nucleotide sequence comprises at least one TNA nucleoside which is linked to an adjacent nucleoside by a linkage different than a PS internucleoside linkage.

74. The antisense gapmer oligonucleotide according to embodiment 73, wherein the TNA nucleoside is linked to an adjacent nucleoside by a PO internucleoside linkage.

75. The antisense gapmer oligonucleotide according to any one of embodiments 73 and 74, wherein the contiguous nucleotide sequence of formula 5′-F-G-F′-3′ (I) has a length of 12 to 32 nucleosides, such as from 12 to 28 nucleosides, such as from 12 to 26 nucleosides, such as from 14 to 26 nucleosides, such as from 14 to 24 nucleosides, such as from 14 to 22 nucleosides, such as from 16 to 22 nucleosides, such as from 16 to 20 nucleosides.

76. The antisense gapmer oligonucleotide according to any one of embodiments 73 to 75, wherein the contiguous nucleotide sequence is of the formula

• • F_1-15-G_3-18-F′_1-15 (II), such as F_1-15-G_3-18-F′_1-12 (IIa) or F_1-12-G_3-18-F′_1-15 (IIb);
  • F_1-12-G_3-18-F′_1-12 (III), such as F_1-12-G_3-18-F′_1-9 (IIIa), or F_1-9-G_3-18-F′_1-12 (IIIb);
  • F_1-12-G_4-16-F′_1-12 (IV), such as F_1-12-G_4-16-F′_1-9 (IVa), or F_1-9-G_4-16-F′_1-12 (IVb); or
  • F_3-12-G_4-10-F′_3-12 (V), such as F_3-12-G_4-10-F′_3-9 (Va) or F_3-9-G_4-10-F′_3-12 (Vb), wherein the numeric ranges represent the number of linked nucleosides in F, G and F′, respectively.

77. The antisense gapmer according to any one of embodiments 73 to 76, further comprising the features of any one of embodiments 1 to 72.

78. The antisense gapmer oligonucleotide according to any one of embodiments 65 to 77, wherein the contiguous nucleotide sequence, optionally of formula IV, such as IVa, has a length of at least 16 nucleosides and

• • (a) the 5′-most nucleosides in F and the nucleosides in F′ are independently 3, 4 or 5 high-affinity sugar-modified nucleosides,
  • (b) the remaining nucleosides in F are TNA nucleosides, and
  • (c) all nucleosides in G are DNA nucleosides.

79. The antisense gapmer oligonucleotide according to embodiment 78, wherein G comprises at most 10 contiguous DNA nucleosides, such as 9, 8, 7, 6, 5, or 4 contiguous DNA nucleosides.

80. The antisense gapmer oligonucleotide according to any one of embodiments 65 to 77, wherein the contiguous nucleotide sequence, optionally of formula IV, has a length of at least 16 nucleosides and

• • (a) F and F′ each independently consists of 3, 4, or 5 nucleosides,
  • (b) wherein at least one of the nucleosides in F and F′ is a TNA nucleoside and the remaining nucleosides are high-affinity sugar-modified nucleosides,
  • (c) all nucleosides in G are DNA nucleosides.

81. The antisense gapmer oligonucleotide of embodiment 80, wherein all nucleosides in F and F′ are TNA nucleosides.

82. The antisense gapmer oligonucleotide according to any one of embodiments 66 to 77, wherein

• • (a) each TNA nucleoside in F and F′ is linked to an adjacent nucleoside by a PO internucleoside linkage, or
  • (b) except for the 5′-most TNA nucleoside in F and the 3′-most TNA nucleoside in F′, each TNA nucleoside in F and F′ is linked to an adjacent nucleoside by a PO internucleoside linkage.

83. The antisense gapmer oligonucleotide according to any one of embodiments 65 to 77, wherein the contiguous nucleotide sequence of formula IV has a length of at least 16 nucleosides and

• • (a) F and F′ each independently comprises or consists of 3, 4, or 5 linked high-affinity sugar-modified nucleosides and does not comprise any TNA nucleoside, and
  • (b) the second, third, fourth or fifth 5′-most nucleoside in G is a TNA nucleoside, such as a TNA (PO) nucleoside, and the remaining nucleosides in G are DNA nucleosides.

84. The antisense gapmer oligonucleotide according to any one of embodiments 78 to 83, wherein the high-affinity sugar modified nucleosides are selected from the sugar-modified nucleosides in embodiment 43.

85. The antisense oligonucleotide according to any one of the preceding embodiments, wherein the antisense oligonucleotide is a single-stranded antisense oligonucleotide.

86. A conjugate comprising the antisense gapmer oligonucleotide according to any one of the preceding embodiments and at least one conjugate moiety covalently attached to said oligonucleotide, optionally via a linker.

87. The conjugate according to embodiment 86, wherein the conjugate moiety is selected from carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins, vitamins, viral proteins and combinations thereof.

88. The conjugate according to embodiment 86 or 87, wherein the conjugate moiety facilitates delivery across the blood brain barrier.

89. A pharmaceutically acceptable salt of the antisense gapmer oligonucleotide according to any one of embodiments 1 to 85, or the conjugate according to any one of embodiments 86 to 88.

90. A pharmaceutical composition comprising the antisense gapmer oligonucleotide according to any one of embodiments 1 to 85, or the conjugate according to any one of embodiments 86 to 88, the pharmaceutically acceptable salt according to embodiment 89, and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

91. An antisense oligonucleotide according to any one of embodiments 1 to 85, a conjugate according to any one of embodiments 86 to 88, the pharmaceutically acceptable salt according to embodiment 89, or a pharmaceutical composition according to embodiment 90, for use as a medicament.

92. A method of preparing a modified version of a parent antisense gapmer oligonucleotide, wherein the parent antisense gapmer comprises a contiguous nucleotide sequence of formula 5′ F-G-F′ 3′ (I) which is capable of recruiting RNase H, wherein G is a gap region of 5 to 18 linked DNA nucleosides and each of F and F′ is a flanking region of up to 8 linked nucleosides which independently comprises or consists of 1 to 8 sugar-modified nucleosides other than TNA nucleosides, and wherein, in the modified version, at least one nucleoside in F, F′, and/or G of the parent antisense gapmer oligonucleotide has been replaced with a TNA nucleoside linked to an adjacent nucleoside by a PO internucleoside linkage,

• • the method comprising the step of manufacturing the modified antisense gapmer oligonucleotide by reacting nucleotide units to form covalently linked contiguous nucleotide units comprised in the oligonucleotide, wherein at least one of the nucleotide units comprises a TNA nucleoside, and,
  • optionally purifying or isolating the modified antisense gapmer oligonucleotide.

93. The method according to embodiment 92, wherein the modified antisense gapmer oligonucleotide has a reduced toxicity, optionally hepatotoxicity, as compared to the parent antisense gapmer oligonucleotide.

94. The method according to any one of embodiments 92 and 93, wherein the modified antisense gapmer oligonucleotide is less toxic to HepG2 cells than the parent antisense gapmer oligonucleotide, optionally as determined by a Caspase 3/7 assay.

95. The method according to any one of embodiments 92 to 94, wherein the modified antisense gapmer oligonucleotide has an increased exonuclease resistance as compared to the parent antisense gapmer oligonucleotide.

96. The method according to any one of embodiments 92 to 95, wherein the modified antisense gapmer oligonucleotide has an increased endonuclease resistance as compared to the parent antisense gapmer oligonucleotide.

97. The method according to any one of embodiments 92 to 96, wherein the parent antisense gapmer oligonucleotide is an LNA gapmer or MOE gapmer, optionally wherein all internucleoside linkages are phosphorothioate linkages.

98. The method according to any one of embodiments 92 to 97, wherein the nucleotide units are nucleoside phosphoramidites.

99. The method according to any one of embodiments 92 to 98, wherein the modified antisense gapmer oligonucleotide comprises the features of any one of embodiments 1 to 84.

100. An antisense gapmer oligonucleotide obtained or obtainable by the method according to any one of embodiments 92 to 99.

101. Use of a TNA nucleotide in the preparation of an antisense gapmer oligonucleotide according to any one of embodiments 1 to 85 or the conjugate of any one of embodiments 86 to 88.

EXAMPLES

The following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated the generic application of those specific embodiments. For example, disclosure of an oligonucleotide having TNA (PO) for one target provides reasonable support for other targets.

Example 1: Oligonucleotide Synthesis Using TNA Modification

Oligonucleotides were synthesized either on a MerMade 12 or on a Mermade 192 automated DNA synthesizer by BioAutomation. Syntheses were conducted on a 1 μmol scale using a controlled pore glass support (500 Å) bearing a universal linker.

For the Mermade 12 synthesis: standard cycle procedures for the coupling of DNA, LNA and MOE phosphoramidites: DMT deprotection was performed with 3% (w/v) trichloroacetic acid in CH₂Cl₂ in six applications of 230 μL for 70 sec. The respective phosphoramidites were coupled three times with 95 μL of 0.1M solutions in acetonitrile (or acetonitrile/CH₂Cl_{2 1:1} for the LNA-^mC building block) and 110 μL of a 0.3 M solution of 5-Benzylthio-1-H-tetrazole in anhydrous acetonitrile as an activator and a coupling time of 180 sec. Freshly prepared TNA phosphoramidites were coupled three times with 95 μL of 0.1M solution in acetonitrile and 110 μL of a 0.3 M solution of 5-Benzylthio-1-H-tetrazole in anhydrous acetonitrile as an activator and a coupling time of 360 sec. Sulfurization was performed using a 0.1M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile/pyridine: 1/1 in two applications of 200 μL for 80 sec. Oxidation was performed using a 0.02M 12 in THF/pyr/H₂O: 88/10/2 in two applications for 80 sec. Capping was performed using THF/lutidine/AC₂O: 8/1/1 (CapA, 125 μL) and THF/N-methylimidazole: 84/16 (CapB, 125 μL) two times for 85 sec. After the synthesis, the CPG was then transferred carefully into a 4 mL vial where 1 mL of 25% NH₄OH was added and left for 16 hours at 55° C. Crude DMT-on oligonucleotides were purified either using a solid-phase extraction cartridge (Oasis HLB 6 cc extraction cartridges from Waters) or by preparative reversed-phase HPLC (RP-HPLC) purification (C18 column, NH₄OAc/CH₃CN buffer system) followed by DMT removal with 80% aqueous acetic acid. Following HPLC purification, oligonucleotides were desalted using HiPrep 26/10 desalting column on ÄKTA pure 25 and lyophilized. Oligonucleotides were characterized by reversed phase liquid chromatography coupled to high-resolution electrospray mass spectrometry.

For the Mermade 192 synthesis: standard cycle procedures for the coupling of DNA, LNA, MOE and TNA phosphoramidites: DMT deprotection was performed with 3% (w/V) trichloroacetic acid in CH₂Cl₂ in three applications of 180 μL for 75 sec. The respective DNA and LNA phosphoramidites were coupled three times with 95 μL of 0.1M solutions in acetonitrile (or acetonitrile/CH₂Cl_{2 1:1} for the LNA-^MeC building block) and 110 μL of a 0.285M solution of 5-Benzylthio-1-H-tetrazole in anhydrous acetonitrile as an activator and a coupling time of 205 sec. Freshly prepared TNA phosphoramidites were coupled two times with 95 μL of 0.1M solution in acetonitrile/DMF 95:5 and 110 μL of a 0.285M solution of 5-Benzylthio-1-H-tetrazole in anhydrous acetonitrile/DMF 95:5 as an activator and a coupling time of 385 sec. Sulfurization was performed using a 0.1M solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile/pyridine: 1/1 in two applications of 190 μL for 385 sec. Oxidation was performed using a 0.02M 12 in THF/pyr/H20: 88/10/2 in two applications of 160 μL for 78 sec. Capping was performed using THF/Lutidine/Ac20 8:1:1 (CapA, 125 μL) and THF/N-methylimidazole: 84/16 (CapB, 125 μL) two times for 70 sec. After the synthesis, the oligonucleotides were cleaved off the CPG in 3 portions of 200 μl using 25% NH₄OH. After every step, the CPG was allowed to stand at RT for 10 min. After the DWP was sealed and the solutions were left for 15 hr at 55° C. The crude DMT-on oligonucleotides were then purified and final DMT cleaved by cartridge (Agilent TOP-DNA Tubes, 150 mg Resin/tube) using aqueous NH₄Cl Buffers, acetonitrile and 5% TFA in Water.

The TNA phosphoramidites were synthesized as described: “Synthesis of Threose Nucleic Acid (TNA) Phosphoramidite Monomers and Oligonucleotide Polymers, Current Protocols in Nucleic Acid Chemistry, 4.51.1-4.51.26, 2012”. All other reagents were purchased from Sigma Aldrich.

Tables 1-5 show molecules prepared following the above procedure, using the designations shown below (which also apply to molecules shown in Tables 7 to 14).

• • A, G, ^mC, T (in bold): represent TNA modification
  • A, G, ^mC, I (underline): represent MOE modification
  • A, G, ^mC, T represent LNA nucleotides
  • a, g, c, ^mC, t represent DNA nucleotides
  • represents phosphodiester linkages
  • * represents phosphorodithioate linkages
  • all other linkages are phosphorothioate linkages.

TABLE 1
Synthesized molecules containing TNA(PO) moieties (targeting
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)).

		Calculated	Found
		mass	mass
CMP ID NO	Sequence	(g/mol)	(g/mol)

CONTROL #1	GAGttacttgccaAmCT	5279.24	5280.9
#1	GAGoTtacttgccaAmCT	5249.15	5250.6
#2	GAGoToTacttgccaAmCT	5219.06	5219.1
#3	GAGoToToActtgccaAmCT	5188.97	5189.2
#4	GAGoToToAomCttgccaAmCT	5172.9	5173.8
#5	GAGoToToAomCoTtgccaAmCT	5142.81	5142.6
#6	GAoGttacttgccaAmCT	5137.07	5136.3
#7	GoAoGttacttgccaAmCT	5121	5121
#8	GoAoGoTtacttgccaAmCT	5090.91	5090.7
#9	GoAoGoToTacttgccaAmCT	5060.82	5060.7
#10	GoAoGoToToActtgccaAmCT	5030.73	5030.7
#11	GoAoGoToToAomCttgccaAmCT	5014.66	5013.9
#12	GoAoGoToToAomCoTtgccaAmCT	4984.57	4984.2
#13	GoAoGoToToActtgccaamCT	5002.72	5002.1
#14	GoAoGoToToActtgccaacT	4960.68	4959.9
#15	GoAoGoToToAomCttgccaamCT	4986.65	4986.6
#16	GoAoGoToToAomCttgccaacT	4944.61	4944.3
#17	GoAoGoToToAomCoTtgccaamCT	4956.56	4956
#18	GoAoGoToToAomCoTtgccaacT	4914.52	4913.7
#19	GoAGttacttgccaAmCoT	5163.04	5163
#20	GoAGttacttgccaAomCoT	5104.94	5105.7
#21	GoAoGttacttgccaAomCoT	5046.84	5047.2
#22	GAoGttacttgccaoAomCT	5146.97	5147.4
#23	GAoGottacttgccaoAomCT	5130.91	5130.9
#24	GAoGttacttgccaAomCT	5163.04	5162.7
#25	GAoGottacttgccaAomCT	5146.97	5146.8
#26	GAoGottacttgccaAomCoT	5130.91	5130.3
#27	GoAGttacttgccaAomCT	5163.04	5162.7
#28	GoAoGttacttgccaAomCT	5146.97	5147.7
#29	GoAoGttacttgccaAmCT	5205.08	5205.6
#30	GAoGottacttgccaAmCT	5205.08	5206.2
#31	GAGoTotacttgccaAmCT	5233.09	5233.2
#32	GAGtoToacttgccaAmCT	5233.09	5233.8
#33	GAGttoAocttgccaAmCT	5233.09	5232.9
#34	GAGttaomCottgccaAmCT	5247.11	5248.2
#35	GAGttacoTotgccaAmCT	5233.09	5234.4
#36	GAGttactoTogccaAmCT	5233.09	5234.4
#37	GAGttacttoGoccaAmCT	5233.09	5232.6
#38	GAGttacttgomCocaAmCT	5247.11	5248.2
#39	GAGttacttgcomCoaAmCT	5247.11	5247
#40	GAGttacttgccoAoAmCT	5233.09	5233.5
#41	GAGttacttgccaoAomCT	5205.08	5207.1
#42	GAGttacttgccaAomCoT	5205.08	5205.6
#43	GAGttacttgccaAmCoT	5221.14	5221.8
#44	GoAGttacttgccaAmCT	5221.14	5220.9
#45	GAoGttacttgccaAmCT	5221.14	5221.5
#46	GAGoTtacttgccaAmCT	5249.15	5249.7
#47	GAGtoTacttgccaAmCT	5249.15	5249.4
#48	GAGttoActtgccaAmCT	5249.15	5250.6
#49	GAGttaomCttgccaAmCT	5263.18	5263.8
#50	GAGttacoTtgccaAmCT	5249.15	5249.7
#51	GAGttactoTgccaAmCT	5249.15	5251.2
#52	GAGttacttoGccaAmCT	5249.15	5249.7
#53	GAGttacttgomCcaAmCT	5263.18	5264.1
#54	GAGttacttgcomCaAmCT	5263.18	5264.4
#55	GAGttacttgccoAAmCT	5249.15	5250.6
#56	GAGttacttgccaoAmCT	5221.14	5222.4
#57	GAoGttacttgccaAmCT	5221.14	5221.5
#58	GAGottacttgccaAmCT	5221.14	5221.2
#59	GAGTotacttgccaAmCT	5249.15	5249.1
#60	GAGtToacttgccaAmCT	5249.15	5249.7
#61	GAGttAocttgccaAmCT	5249.15	5249.4
#62	GAGttamCottgccaAmCT	5263.18	5263.8
#63	GAGttacTotgccaAmCT	5249.15	5250
#64	GAGttactTogccaAmCT	5249.15	5249.7
#65	GAGttacttGoccaAmCT	5249.15	5250.6
#66	GAGttacttgmCocaAmCT	5263.18	5264.4
#67	GAGttacttgcmCoaAmCT	5263.18	5264.7
#68	GAGttacttgccAoAmCT	5249.15	5249.1
#69	GAGttacttgccaAomCT	5221.14	5220.9
#70	GAGttacttgccaAmCoT	5221.14	5222.7
CONTROL #2	GmCmCAGgctggttatgAmCTmCA	7235.11	7235.6
#71	GmCmCAoGgctggttatgAmCTmCA	6778.52	6780
#72	GmCmCoAoGgctggttatgAmCTmCA	6762.45	6763.2
#73	GmComCoAoGgctggttatgAmCTmCA	6746.39	6747.2
#74	GomComCoAoGgctggttatgAmCTmCA	6730.32	6730.8
#75	GomComCoAoGoGctggttatgAmCTmCA	6700.23	6700.4
#76	GomComCoAoGoGomCtggttatgAmCTmCA	6684.17	6685.6
#77	GomComCoAoGoGomCoTggttatgAmCTmCA	6654.07	6654.8
#78	GomComCoAoGoGomCoToGgttatgAmCTmCA	6623.98	6624
#79	GomComCoAoGoGomCoToGoGttatg	6593.89	6594
	AmCTmCA
#80	GmCmCAGoGctggttatgAmCTmCA	7205.02	7205.5
#81	GmCmCAGoGoctggttatgAmCTmCA	7188.95	7188.9
#82	GmCmCAGoGomCtggttatgAmCTmCA	7188.95	7189.6
#83	GmCmCAGoGomCotggttatgAmCTmCA	7172.89	7173.1
#84	GmCmCAGoGomCoTggttatgAmCTmCA	7158.86	7158.6
#85	GmCmCAGoGomCoToggttatgAmCTmCA	7142.8	7143
#86	GmCmCAGoGomCoToGgttatgAmCTmCA	7128.77	7129
#87	GmCmCAGoGomCoToGogttatgAmCTmCA	7112.7	7112.5
#88	GmCmCAGoGomCoToGoGttatgAmCTmCA	7098.68	7099.1
#89	GmCmCAGoGomCoToGoGottatgAmCTmCA	7082.61	7082.4
#90	GmCmCAGoGomCoToGoGoTtatgAmCTmCA	7068.58	7068.9
#91	GomCmCAGgctggttatgAmCTmCoA	7026.77	7028.4
#92	GomCmCAGgctggttatgAmCTomCoA	6922.6	6922
#93	GomComCAGgctggttatgAmCTomCoA	6818.43	6819.2
#94	GomComCAGoGctggttatgAmCTomCoA	6788.34	6788.4
#95	GomComCAGoGomCtggttatgAmCTomCoA	6772.27	6772.8
#96	GomComCAGoGomCoTggttatgAmCTomCoA	6742.18	6742.8
#97	GomComCAGoGomCoToGgttatgAmCTomCoA	6712.09	6712
#98	GomComCAGoGomCoToGoGttatgAmCT	6681.99	6682.4
	omCoA
#99	GomComCoAoGgctggttatgAomCoTomCoA	6625.53	6225.6
#100	GmComCoAoGgctggttatgAomCoTomCA	6257.67	6258.8
#101	GmCmCAGgctggttatgAomCoTomCoA	6730.32	6731.1
#102	GmCmCAGgctggttatGoAomCoTomCoA	6700.23	6700.4
#103	GmCmCAGgctggttaToGoAomCoTomCoA	6670.14	6670.5
#104	GmCmCAGgctggttAoToGoAomCoTomCoA	6640.05	6640.7
#105	GmCmCAGgctggttatgAmCTmCoA	7042.84	7042.4
#106	GmCmCAGgctggttatgAmCTomCoA	7026.77	7026
#107	GomCmCAGgctggttatgAmCTomCoA	7026.77	7026.8
#108	GmComCoAGgctggttatgAmCoTomCA	6994.64	6996
#109	GmComCoAGgctggttatgAomCoTmCA	6994.64	6994
#110	GmCmCAGoGoctggttatoGoAmCTmCA	7142.8	7142
#111	GmComCoAGgctggttatgAomCoTomCA	6890.47	6891.2
#112	GmComCoAoGgctggttatgAomCoTomCA	6786.3	6786.4
#113	GomComCAGgctggttatgAmCTmCA	7114.87	7114.8
#114	GmComCoAGgctggttatgAmCTmCA	7114.87	7114.4
#115	GmCmCoAoGgctggttatgAmCTmCA	7114.87	7116.4
#116	GmCmCAoGogctggttatgAmCTmCA	7114.87	7114.8
#117	GmCmCAGoGoctggttatgAmCTmCA	7188.95	7189.6
#118	GmCmCAGgomCotggttatgAmCTmCA	7202.98	7204.4
#119	GmCmCAGgcoToggttatgAmCTmCA	7188.95	7188.8
#120	GmCmCAGgctoGogttatgAmCTmCA	7188.95	7188.8
#121	GmCmCAGgctgoGottatgAmCTmCA	7188.95	7189.2
#122	GmCmCAGgctggoTotatgAmCTmCA	7188.95	7190
#123	GmCmCAGgctggtoToatgAmCTmCA	7188.95	7188.8
#124	GmCmCAGgctggttoAotgAmCTmCA	7188.95	7189.2
#125	GmCmCAGgctggttaoTogAmCTmCA	7188.95	7189.2
#126	GmCmCAGgctggttatoGoAmCTmCA	7188.95	7190
#127	GmCmCAGgctggttatgoAomCTmCA	7114.87	7114.8
#128	GmCmCAGgctggttatgAomCoTmCA	7114.87	7114.8
#129	GmCmCAGgctggttatgAmCoTomCA	7114.87	7116.4
#130	GmCmCAGgctggttatgAmCTomCoA	7114.87	7115.6
#131	GmCmCAGgctggttatgAmCTmCoA	7130.94	7131.2
#132	GomCmCAGgctggttatgAmCTmCA	7130.94	7130.4
#133	GomCmCAGgctggttatgAmCTmCA	7130.94	7131.6
#134	GmComCAGgctggttatgAmCTmCA	7130.94	7131.2
#135	GmCmCoAGgctggttatgAmCTmCA	7130.94	7131.2
#136	GmCmCAoGgctggttatgAmCTmCA	7130.94	7132
#137	GmCmCAGoGctggttatgAmCTmCA	7205.02	7205.2
#138	GmCmCAGgomCtggttatgAmCTmCA	7219.05	7219.2
#139	GmCmCAGgcoTggttatgAmCTmCA	7205.02	7204.8
#140	GmCmCAGgctoGgttatgAmCTmCA	7205.02	7204.4
#141	GmCmCAGgctgoGttatgAmCTmCA	7205.02	7206
#142	GmCmCAGgctggoTtatgAmCTmCA	7205.02	7204.8
#143	GmCmCAGgctggtoTatgAmCTmCA	7205.02	7204
#144	GmCmCAGgctggttoAtgAmCTmCA	7205.02	7205.2
#145	GmCmCAGgctggttaoTgAmCTmCA	7205.02	7205.2
#146	GmCmCAGgctggttatoGAmCTmCA	7205.02	7206.4
#147	GmCmCAGgctggttatgoAmCTmCA	7130.94	7132
#148	GmCmCAGgctggttatgAomCTmCA	7130.94	7132
#149	GmCmCAGgctggttatgAmCoTmCA	7130.94	7131.2
#150	GmCmCAGgctggttatgAmCTomCA	7130.94	7131.2
CONTROL #3	GGmCATatgcagataaTGTTmC	7230.09	7230
#151	GGmCAToAtgcagataaTGTTmC	7200	7200.8
#152	GGmCAToAoTgcagataaTGTTmC	7169.91	7170.2
#153	GGmCAToAoToGcagataaTGTTmC	7139.82	7140.5
#154	GGmCAToAoToGomCagataaTGTTmC	7123.75	7123.9
#155	GGmCAToAoToGomCoAgataaTGTTmC	7093.66	7093.5
#156	GGmCAoTatgcagataaTGTTmC	6773.5	6774.4
#157	GGmCoAoTatgcagataaTGTTmC	6757.44	6760.4
#158	GGomCoAoTatgcagataaTGTTmC	6741.37	6744
#159	GoGomCoAoTatgcagataaTGTTmC	6725.31	6726.2
#160	GoGomCoAoToAtgcagataaTGTTmC	6695.21	6696.4
#161	GoGomCoAoToAoTgcagataaTGTTmC	6665.12	6664.4
#162	GoGomCoAoToAoToGcagataaTGTTmC	6635.03	6634.4
#163	GoGomCoAoToAoToGomCagataaTGTTmC	6618.96	6618.4
#164	GoGomCoAoToAoToGomCoAgataaTGTTmC	6588.87	6590.4
#165	GoGomCoAoToAoToGomCoAgataatGTTmC	6514.79	6514.4
#166	GoGomCoAoToAoToGomCoAgataatgTTmC	6440.72	6440
#167	GoGomCoAoToAoToGomCoAoGataatGTTmC	6484.7	6484
#168	GoGomCoAoToAoToGomCoAoGataatgTTmC	6410.62	6411.2
#169	GoGmCATatgcagataaTGTTomC	7021.75	7023.1
#170	GoGmCATatgcagataaTGToTomC	6917.58	6917.2
#171	GoGomCATatgcagataaTGToTomC	6813.41	6812.8
#172	GoGomCAToAtgcagataaTGToTomC	6783.32	6782.8
#173	GoGomCAToAoTgcagataaTGToTomC	6753.23	6752.3
#174	GoGomCAToAoToGcagataaTGToTomC	6723.13	6723.3
#175	GoGomCAToAoToGomCagataaTGToTomC	6707.07	6707.6
#176	GoGomCAToAoToGomCoAgataaTGToTomC	6676.98	6676.4
#177	GoGomCoAoTatgcagataaToGoToTomC	6220.53	6220.4
#178	GGomCoAoTatgcagataaToGoToTmC	6252.65	6253.2
#179	GGmCATatgcagataaToGoToTomC	6725.31	6725.6
#180	GGmCATatgcagataAoToGoToTomC	6695.21	6694.9
#181	GGmCATatgcagatAoAoToGoToTomC	6665.12	6665.6
#182	GGmCATatgcagaToAoAoToGoToTomC	6635.03	6635.6
#183	GGmCATatgcagataaTGTTomC	7037.82	7037.6
#184	GGmCATatgcagataaTGToTomC	7021.75	7021.6
#185	GGomCATatgcagataaTGoTTmC	7021.75	7023.6
#186	GGomCoATatgcagataaTGoToTmC	6989.62	6990.4
#187	GGomCoATatgcagataaTGoToTmC	6901.56	6902.4
#188	GGomCoATatgcagataaToGoToTmC	6885.45	6885.6
#189	GGomCoAoTatgcagataaToGoToTmC	6781.28	6780.4
#190	GGmCAToAtgcagataoATGTTmC	7169.91	7171.2
#191	GGmCAToAotgcagataoAoTGTTmC	7137.78	7137.6
CONTROL #4	GGGAGttacttgccaAmCTTG	7208.04	7208.1
#192	GGGAGoTtacttgccaAmCTTG	7177.95	7178.8
#193	GGGAGoToTacttgccaAmCTTG	7147.86	7148.3
#194	GGGAGoToToActtgccaAmCTTG	7117.77	7118
#195	GGGAGoToToAomCttgccaAmCTTG	7101.7	7102
#196	GGGAGoToToAomCoTtgccaAmCTTG	7071.61	7071.4
#197	GGGAGoToToAomCoToTgccaAmCTTG	7041.52	7041.5
#198	GoGoGAGttacttgccaAmCToToG	6791.36	6790.8
#199	GoGoGAGoTtacttgccaAmCToToG	6761.27	6760.8
#200	GoGoGAGoToTacttgccaAmCToToG	6731.18	6730.4
#201	GoGoGAGoToToActtgccaAmCToToG	6701.08	6701.2
#202	GoGoGAGoToToAomCttgccaAmCToToG	6685.02	6685.2
#203	GoGoGAGoToToAomCoTtgccaAmCToToG	6654.93	6655.6
#204	GoGoGoAoGttacttgccaAomCoToToG	6198.47	6198
#205	GGoGoAoGttacttgccaAomCoToTG	6230.6	6229.6
#206	GGGAoGttacttgccaAmCTTG	6751.45	6751.7
#207	GGGoAoGttacttgccaAmCTTG	6735.39	6735.7
#208	GGoGoAoGttacttgccaAmCTTG	6719.32	6719.5
#209	GoGoGoAoGttacttgccaAmCTTG	6703.25	6703.6
#210	GoGoGoAoGoTtacttgccaAmCTTG	6673.16	6673
#211	GoGoGoAoGoToTacttgccaAmCTTG	6643.07	6642.4
#212	GoGoGoAoGoToToActtgccaAmCTTG	6612.98	6612.8
#213	GoGoGoAoGoToToAomCttgccaAmCTTG	6596.91	6596.1
#214	GoGoGoAoGoToToAomCoTttgccaAmCTTG	6566.82	6566.9
#215	GoGoGoAoGoToToAomCoTttgccaamCTTG	6492.74	6492.4
#216	GoGoGoAoGoToToAomCoTttgccaacTTG	6404.64	6403.6
#217	GGGAGttacttgccaAomCoToToG	6703.25	6704.7
#218	GGGAGttacttgcmCoAoAomCoToToG	6657.1	6658.1
CONTROL #5	TGmCmCTttaggattctAGAmCA	7195.08	7195.4
#219	TGmCmCToTtaggattctAGAmCA	7164.99	7165.7
#220	TGmCmCToToTaggattctAGAmCA	7134.9	7135.5
#221	TGmCmCToToToAggattctAGAmCA	7104.81	7105.4
#222	TGmCmCToToToAoGgattctAGAmCA	7074.71	7075.7
#223	TGmCmCToToToAoGoGattctAGAmCA	7044.62	7045.7
#224	TGmCmCToToToAoGoGoAttctAGAmCA	7014.53	7015.1
#225	ToGomComCoTttaggattctAoGoAomCoA	6185.51	6184.4
#226	TGomComCoTttaggattctAoGoAomCA	6217.64	6217.6
#227	TGmCmCoTttaggattctAGAmCA	6738.49	6738.3
#228	TGmComCoTttaggattctAGAmCA	6722.43	6722.6
#229	TGomComCoTttaggattctAGAmCA	6706.36	6705.7
#230	ToGomComCoTttaggattctAGAmCA	6690.3	6690.5
#231	ToGomComCoToTtaggattctAGAmCA	6660.2	6660
#232	ToGomComCoToToTaggattctAGAmCA	6630.11	6630
#233	ToGomComCoToToToAggattctAGAmCA	6600.02	6599.8
#234	ToGomComCoToToToAoGgattctAGAmCA	6569.93	6569
#235	ToGomComCoToToToAoGoGattctAGAmCA	6539.83	6539.1
#236	ToGomComCoToToToAoGoGattctaGAmCA	6465.76	6466.4
#237	ToGomComCoToToToAoGoGattctagAmCA	6391.69	6392.4
#238	TGmCmCTttaggattctAoGoAomCoA	6690.3	6690.5
#239	TGmCmCTttaggattcToAoGoAomCoA	6660.2	6660.7
#240	TGmCmCTttaggattmCoToAoGoAomCoA	6644.14	6642.8
CONTROL #6	mCmCAGGctggttatgamCTmCAG	7235.11	7234
#241	mCmCAGoGctggttatgamCTmCAG	6778.52	6778
#242	mCmCAoGoGctggttatgamCTmCAG	6762.45	6762
#243	mCmCoAoGoGctggttatgamCTmCAG	6746.39	6746.4
#244	mComCoAoGoGctggttatgamCTmCAG	6730.32	6729.2
#245	mComCoAoGoGomCtggttatgamCTmCAG	6714.26	6714
#246	mComCoAoGoGomCoTggttatgamCTmCAG	6684.17	6683.2
#247	mComCoAoGoGomCoToGgttatgamCTmCAG	6654.04	6654
#248	mComCoAoGoGomCoToGoGttatgamCTmCAG	6623.98	6624
CONTROL #7	TTATmCaattcaccaaGGAGmC	7159.06	7158
#249	TTATomCaattcaccaaGGAGmC	6702.47	6702.4
#250	TTAoTomCaattcaccaaGGAGmC	6686.4	6685.6
#251	TTOAoTomCaattcaccaaGGAGmC	6670.34	6672.4
#252	ToToAoTomCaattcaccaaGGAGmC	6654.27	6654
#253	ToToAoTomCoAattcaccaaGGAGmC	6624.18	6624
#254	ToToAoTomCoAoAttcaccaaGGAGmC	6594.09	6593.2
#255	ToToAoTomCoAoAoTtcaccaaGGAGmC	6564	6563.6
#256	ToToAoTomCoAoAoToTcaccaaGGAGmC	6533.9	6533.2
#257	ToToAoTomCoAoAoToTomCaccaaGGAGmC	6517.84	6518
CONTROL #8	ATGGAggtatgacatATAAT	7250.09	7250.4
#258	ATGGoAggtatgacatATAAT	6793.5	6792.4
#259	ATGoGoAggtatgacatATAAT	6777.43	6776.4
#260	AToGoGoAggtatgacatATAAT	6761.37	6761.2
#261	AoToGoGoAggtatgacatATAAT	6745.3	6744.4
#262	AoToGoGoAoGgtatgacatATAAT	6715.21	6713.6
#263	AoToGoGoAoGoGtatgacatATAAT	6685.12	6684.8
#264	AoToGoGoAoGoGoTatgacatATAAT	6655.03	6654
#265	AoToGoGoAoGoGoToAtgacatATAAT	6624.93	6624
#266	AoToGoGoAoGoGoToAoTgacatATAAT	6594.84	6594.4
CMP ID NO = Compound ID number.

We have designed two libraries of antisense oligonucleotides directed against human MALAT1 (Gencode: ENSG00000251562). Compounds were 20-nucleotide long, with two different sugar patterns: Design A: TTTTTddddddddddTTTTT, where T is TNA and d is DNA, and with the backbone pattern SOOOssssssssssSOOOS, where S corresponds to phosphorothioate, and O to phosphodiester. Design B: MMMMMTTTTTTddddMMMMM, where M is MOE, T is TNA and d is DNA, and with the backbone pattern ssssoooooossssssSSS, where S corresponds to phosphorothioate, and O to phosphodiester. The compounds were tested at two different concentrations (5 & 25 μM) and the 18 most potent oligonucleotides for Design A (#267-284) and the 19 most potent oligonucleotides for Design B (#285-303) were selected for full dose response determinations.

TABLE 2
Selected compounds from the two libraries synthesized
containing TNA PO (targeting MALAT1).

CMP			Calculated	Found
ID			mass	mass
NO	Design	Sequence	(g/mol)	(g/mol)

#267	Design A	GToToAoTgcttattcccmCoAoAoTG	6140.55	6140
#268	Design A	mCTomCoAoGcctttatcacToCoAoGmC	6112.58	6112
#269	Design A	AAomCoAoAatttccttagToToGoGmC	6187.61	6186.9
#270	Design A	TAomCoAomCcagtccttttAoGoToAG	6163.59	6163
#271	Design A	AToGomCoTcatcactttaToGoAoAG	6173.59	6173
#272	Design A	AAoAoAoGgcttagmCgccmCoAomComCT	6195.64	6195.2
#273	Design A	AAoGoAoGaaccacacacToAomComCA	6153.62	6153.2
#274	Design A	mCAoTomCoTcaacctcmCgtmCoAoToGT	6112.58	6112
#275	Design A	AAoGoGoTctcatacactmCoAomCoTA	6141.59	6141
#276	Design A	GToAomCoTatcccatcacToGoAoAG	6143.56	6142.9
#277	Design A	GAomComComCctgactttctGoGoAoAA	6187.62	6187.1
#278	Design A	mCAoGomCoGgtacactcctTomCoToCT	6128.58	6128
#279	Design A	GmComCoAoAtatttgccccTomComComCmC	6101.6	6101.1
#280	Design A	TGomCoAoTttacttgccaAomCoAoGA	6172.6	6172.1
#281	Design A	AAoAoGoAgtaactaccaGomComCoAT	6199.64	6199.1
#282	Design A	AAomCoAoGgtcatctattmCoAomCoAA	6179.64	6179.1
#283	Design A	TGoToAomCattttgccctToAoGomCT	6145.56	6145.1
#284	Design A	GmComCoAoAgcactcatatGomCoAoAT	6180.63	6180.1
#285	Design B	GTTAToGomCoToToAoTtcccmCAATG	6951.47	6950.8
#286	Design B	GTGTAoAoToToAomComCttttAmCTmCT	6969.52	6968.9
#287	Design B	mCmCTTmCoAoGoAoGoAoTtcaaTGmCTA	6997.55	6996.8
#288	Design B	GmCmCAAoToAoToToToGccccTmCmCmCmC	6898.49	6897.8
#289	Design B	TGmCTAoAoAomCoAoAoAtttcmCTTAG	6982.53	6981.9
#290	Design B	AAmCAAoAoToToTomComCttagTTGGmC	7012.56	7011.8
#291	Design B	TmCAGToGomCoToAoToTttatmCmCAAT	6978.53	6977.8
#292	Design B	TGTAmCoAoToToToToGccctTAGmCT	6942.45	6941.8
#293	Design B	TTGmCToGoAoAoAoToTgtctmCAATT	6990.5	6989.9
#294	Design B	TTGAAoGomCoAoToAomCcttaAmCATmC	6981.55	6980.9
#295	Design B	mCTmCmCAoAoToAomCoToTgtctTAGmCT	6953.52	6952.8
#296	Design B	GmCmCTToAoAoAoGoToTacatTmCGTT	6989.52	6988.8
#297	Design B	AAGGTomCoTomCoAoToAcactmCAmCTA	6966.54	6965.9
#298	Design B	GTAmCToAoTomComComCoAtcacTGAAG	6982.53	6981.8
#299	Design B	ATGmCAoAoGoToToAoAacttATmCTG	7008.53	7007.8
#300	Design B	TGmCmCTomCoToTomCoAoTtgtaTTTmCT	6950.51	6949.8
#301	Design B	mCATmCAoAoGoGomCoAomCtgatmCAmCTT	7010.59	7009.9
#302	Design B	GmCmCAAoGomCoAomCoTomCatatGmCAAT	7019.6	7018.8
#303	Design B	GGGAGoToToAomCoToTgccaAmCTTG	7041.52	7040.8

TABLE 3
TNA(PO) vs DNA(PO) (targeting MALAT1)

		Calcu-
		lated	Found
CMP		mass	mass
ID NO	Sequence	(g/mol)	(g/mol)

CONTROL	GAGttacttgccaAmCT	5279.24	5280.9
#1
#1	GAGoTtacttgccaAmCT	5249.15	5250.6
#2	GAGoToTacttgccaAmCT	5219.06	5219.1
#3	GAGoToToActtgccaAmCT	5188.97	5189.2
#4	GAGoToToAomCttgccaAmCT	5172.9	5173.8
#5	GAGoToToAomCoTtgccaAmCT	5142.81	5142.6
#304	GAGottacttgccaAmCT	5263.18	5263.9
#305	GAGototacttgccaAmCT	5247.11	5247.9
#306	GAGototoacttgccaAmCT	5231.05	5231.1
#307	GAGototoaocttgccaAmCT	5214.98	5215.5
#308	GAGototoaocottgccaAmCT	5198.92	5199.3

Antisense oligonucleotides targeting Ceramide Synthase 2 (CERS2), Ceramide Synthase 5 (CERS5), and Ceramide Synthase 6 (CERS6) were synthesized and are shown in Table 4. Synthesized molecules containing TNA (PO) and phosphorodithioate (PS₂) moieties are shown in Table 5 (targeting MALAT1).

TABLE 4
Synthesized molecules containing TNA(PO) moieties
(targeting CERS2, CERS5, CERS6).

			Calculated	Found mass
CMP ID NO	Target	Sequence	mass (g/mol)	(g/mol)

CONTROL #9	CERS2	mCTTtggcagaatgAGG	5384.31	5384.4
#309	CERS2	mCTToTggcagaatgAGG	5354.21	5354.7
#310	CERS2	mCTToToGgcagaatgAGG	5324.12	5324.1
#311	CERS2	mCTToToGoGcagaatgAGG	5294.03	5294.1
#312	CERS2	mCTToToGoGomCagaatgAGG	5277.96	5278.2
#313	CERS2	mCTToToGoGomCoAgaatgAGG	5247.87	5248.5
CONTROL #10	CERS2	GmCTttggcagaatGAG	5384.31	5384.8
#314	CERS2	GmCToTtggcagaatGAG	5354.21	5353.2
#315	CERS2	GmCToToTggcagaatGAG	5324.12	5323.8
#316	CERS2	GmCToToToGgcagaatGAG	5294.03	5293.8
#317	CERS2	GmCToToToGoGcagaatGAG	5263.94	5263.5
#318	CERS2	GmCToToToGoGomCagaatGAG	5247.87	5247.6
CONTROL #11	CERS5	AmCatgctttcacagAATT	5912.78	5912.4
#319	CERS5	AmCoAtgctttcacagAATT	5882.68	5882.1
#320	CERS5	AmCoAoTgctttcacagAATT	5852.59	5852
#321	CERS5	AmCoAoToGctttcacagAATT	5822.5	5822.8
#322	CERS5	AmCoAoToGomCtttcacagAATT	5806.43	5805
#323	CERS5	AmCoAoToGomCoTttcacagAATT	5776.34	5776.8
CONTROL #12	CERS6	TTmCAttcacagacagGA	5617.53	5617.2
#324	CERS6	TTmCAoTtcacagacagGA	5587.44	5588
#325	CERS6	TTmCAoToTcacagacagGA	5557.35	5556.8
#326	CERS6	TTmCAoToTomCacagacagGA	5541.28	5540.8
#327	CERS6	TTmCAoToTomCoAcagacagGA	5511.19	5511.6
#328	CERS6	TTmCAoToTomCoAomCagacagGA	5495.12	5494.8
CMP ID NO = Compound ID number.

TABLE 5
Synthesized molecules containing TNA(PO) and
phosphorodithioate (PS2 )moieties (targeting MALAT1).

		Calculated	Found
		mass	mass
CMP ID NO	Sequence	(g/mol)	(g/mol)

CONTROL #1	GAGttacttgccaAmCT	5279.24	5280
#31	GAGoTotacttgccaAmCT	5233.09	5234.7
#32	GAGtoToacttgccaAmCT	5233.09	5235
#329	GAGoToToacttgccaAmCT	5202.99	5204.1
#330	GAGoTotacttgccoAoAmCT	5186.93	5188.8
#331	G*AGoTotacttgccaAmC*T	5265.22	5267.6
#332	G*AGtoToacttgccaAmC*T	5265.22	5267.1
#333	G*AGoToToacttgccaAmC*T	5235.13	5237.1
#334	G*AGoTotacttgccoAoAmC*T	5219.06	5219.1
CONTROL #2	GmCmCAGgctggttatgAmCTmCA	7235.11	7236.4
#81	GmCmCAGoGoctggttatgAmCTmCA	7188.95	7191.6
#118	GmCmCAGgomCotggttatgAmCTmCA	7202.98	7206
#83	GmCmCAGoGomCotggttatgAmCTmCA	7172.89	7176
#110	GmCmCAGoGoctggttatoGoAmCTmCA	7142.8	7144.4
#335	G*mCmCAGoGoctggttatgAmCTC*A	7221.08	7222.9
#336	G*mCmCAGgomCotggttatgAmCTmC*A	7235.11	7237.6
#337	G*mCmCAGoGomCotggttatgAmCTC*A	7205.02	7206.4
#338	G*mCmCAGoGoctggttatoGoAmCTC*A	7174.93	7175.2
CONTROL #3	GGmCATatgcagataaTGTTmC	7230.09	7232.8
#339	GGmCAToAotgcagataaTGTTmC	7183.94	7184.8
#340	GGmCATaoTogcagataaTGTTmC	7183.94	7186
#341	GGmCAToAoTogcagataaTGTTmC	7153.84	7156.4
#191	GGmCAToAotgcagataoAoTGTTmC	7137.78	7137.6
#342	G*GmCAToAotgcagataaTGTT*mC	7216.07	7218
#343	G*GmCATaoTogcagataaTGTT*mC	7216.07	7217.6
#344	G*GmCAToAoTogcagataaTGTT*mC	7185.98	7187.6
#345	G*GmCAToAotgcagataoAoTGTT*mC	7169.91	7172.5
CMP ID NO = Compound ID number.

Further details on the molecules in Tables 1-5 are set out in Table 6, in which the structure of each synthesized molecule is defined by the hierarchical editing language for macromolecules (HELM) (for details, see Zhang et al., Chem. Inf. Model. 2012; 52 (10): 2796-2806). In addition, the SEQ ID NO of the nucleobase sequence upon which each respective synthesized molecule is based is indicated. The following HELM annotation keys are used:

• • [LR](G) is a beta-D-oxy-LNA guanine nucleoside,
  • [LR](T) is a beta-D-oxy-LNA thymine nucleoside,
  • [LR](A) is a beta-D-oxy-LNA adenine nucleoside,
  • [LR]([5meC] is a beta-D-oxy-LNA 5-methyl cytosine nucleoside,
  • [dR](G) is a DNA guanine nucleoside,
  • [dR](T) is a DNA thymine nucleoside,
  • [dR](A) is a DNA adenine nucleoside,
  • [dR](C) is a DNA cytosine nucleoside,
  • [dR]([5meC]) is a DNA 5-methyl cytosine nucleoside,
  • [MOE]([5meC]) is a 2′O-MOE [2′O-(2-methoxyethyl)]5-methyl cytidine nucleoside
  • [MOE](A) is a 2′O-MOE [2′O-(2-methoxyethyl)]adenine nucleoside
  • [MOE](T) is a 2′-O-MOE [2′O-(2-methoxyethyl)]thymine nucleoside
  • [MOE](G) is a 2′-O-MOE [2′O-(2-methoxyethyl)]guanine nucleoside
  • [TNA]([5meC]) is a α-L-threose nucleic acid 5-methyl cytidine nucleoside
  • [TNA](A) is a α-L-threose nucleic acid adenine nucleoside
  • [TNA](T) is a α-L-threose nucleic acid thymine nucleoside
  • [TNA](G) is a α-L-threose nucleic acid guanine nucleoside
  • [sP] is a phosphorothioate internucleoside linkage
  • [PS₂] is a phosphorodithioate internucleoside linkage
  • P is a phosphodiester internucleoside linkage

Further information as well as open-source tools for HELM can be found at the internet addresses www.pistoiaalliance.org/helm-tools/(accessed on 26 Aug. 2022).

TABLE 6
Synthesized molecules in HELM annotations

CMP ID	SEQ ID
NO	NO	Base sequence 5′-3′	HELM
CONTROL	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[SP].[LR](G)[sP].[dR](T)[P].
#1			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[SP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#1	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[TNA](T)[sP].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#2	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[TNA](T)P.[TNA]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#3	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#4	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)P.[TNA]([5meC])[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#5	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#6	1	GAGTTACTTGCCAACT	[TNA](G)[sP].[TNA](A)P.[TNA](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#7	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR].(C)[sP].
			[dRI(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#8	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)[sP].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#9	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#10	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)
			[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#11	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)P.[TNA]([5meC])[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#12	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#13	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)
			[sP].[dR](A)[sP].[LR]([5meC])[sP].[LR](T)
#14	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)
			[sP].[dR](A)[sP].[dR](C)[sP].[LR](T)
#15	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)P.[TNA]([5meC])[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[dR](A)[sP].[LR]([5meC])[sP].[LR](T)
#16	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)P.[TNA]([5meC])[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[dR](A)[sP].[dR](C)[sP].[LR](T)
#17	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[dR](A)[sP].[LR]([5meC])[sP].[LR](T)
#18	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA]
			(T)P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)[P].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			((A)[sP].[dR](A)[sP].[dR](C)[sP].[LR](T)
#19	1	GAGTTACTTGCCAACT	[TNA](G)P.[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].[d
			R(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])P.[TNA](T)
#20	1	GAGTTACTTGCCAACT	[TNA](G)P.[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)P.[TNA]([5meC])P.[TNA](T)
#21	1	GAGTTACTTGCCAACT	[TNA](G)P.[TNA](A)P.[LR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)P.[TNA]([5meC])P.[TNA](T)
#22	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)P.[TNA](G)[sP].[dR](T)[P].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)P.[TNA](A)P.[LR]([5meC])[sP].[LR](T)
#23	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)P.[TNA](G)P.[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)P.[TNA](A)P.[LR]([5meC])[sP].[LR](T)
#24	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)P.[TNA](G)[sP].[dR](T)[sP].[d
			R](T)[SP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)P.[TNA]([5meC])[sP].[LR](T)
#25	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)P.[TNA](G)P.[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)P.[TNA]([5meC])[sP].[LR](T)
#26	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)P.[TNA](G)P.[dR](T)[P].[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)P.[TNA]([5meC])P.[LR](T)
#27	1	GAGTTACTTGCCAACT	[LR](G)P.[TNA](A)[sP].[LR](G)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)P.[TNA]([5meC])[sP].[LR](T)
#28	1	GAGTTACTTGCCAACT	[LR](G)P.[TNA](A)P.[LR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)P.[TNA]([5meC])[sP].[LR](T)
#29	1	GAGTTACTTGCCAACT	[LR](G)P.[TNA](A)P.[LR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A) [P].[LR]([5meC])[sP].[LR](T)
#30	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)P.[TNA](G)P.[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#31	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[TNA](T)P.[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)
			[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#32	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)P.[TN
			A](T)P.[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#33	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[SP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)P.[TNA](A)P.[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]9[5meC])[sP].[LR](T)
#34	1	GÅGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)P.[TNA]([5meC])P.[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[P].[dR](C)[sP].
			[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#35	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[P].[LR](G)[P].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)P.[TNA](T)P.[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#36	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sp].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)P.[TN
			A](T)P.[dR](G)[sP].[dR](C)[sP].[dR](C)[sP][.[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#37	1	GAGTTACTTGCCAACT	[LR](G)[SP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)P.[TNA](G)P.[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#38	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[SP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)P.[TNA]([5meC])P.[dR](C)[sP].
			[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#39	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[SP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)P.[TNA]([5meC])P.
			[dR](A)[sP].[LR](A)[SP].[LR]([5meC])[sP].[LR](T)
#40	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)P.[TN
			A](A)P.[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#41	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[P].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sp].[dR](G)[sp].[dR](C)[sP].[dR](C)[sP].
			[dR](A)P.[TNA](A)P.[LR]([5meC])[P].[LR](T)
#42	1	GAGTTACTTGCCAACT	[LR](G)[SP].[LR](A)[SP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[dR](A)[sP].[LR](A)P.[TNA]([5meC])P.[LR](T)
#43	1	GAGTTACTTGCCAACT	[LR](G)[SP].[LR](A)[SP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[dR](A)[sP].[LR](A)[sP].[LR]([5meC])P.[TNA](T)
#44	1	GAGTTACTTGCCAACT	[LR](G)P.[TNA](A)[sP].[LR](G)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)}
#45	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)P.[TNA](G)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)}
#46	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[TNA](T)[sP].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#47	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)P.[TN
			A](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#48	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)P.[TNA](A)[sP].[dR](C)[sP].[dR](T)[P].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#49	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)P.[TNA]([5meC])[sP].[dR](T)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[s
			P].[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR]
			(T)
#50	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)P.[TNA](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#51	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)P.[TN
			A](T)[sP].[dR](G)[sP].[dR](C)[P].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#52	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)P.[TNA](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#53	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)P.[TNA]([5meC])[sP].[dR](C)
			[sP].[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR]
			(T)
#54	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)P.[TNA]([5meC])
			[sP].[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR]
			(T)
#55	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)P.[TN
			A](A)[sP].[LR](A)[sP].[LR]([5meC])[P].[LR](T)
#56	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[dR](A)P.[TNA](A)[sP].[LR]([5meC])[sP].[LR](T)
#57	1	GAGTTACTTGCCAACT	[LR](G)[sP].[TNA](A)P.[LR](G)[P].[dR](T)[sP].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#58	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[TNA](G)P.[dR](T)[sP].[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#59	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[TNA](T)P.[d
			R](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#60	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[P].[dR](T)[sP].
			[TNA](T)P.[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#61	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[TNA](A)P.[dR](C)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#62	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[P].[dR](A)[sP].[TNA]([5meC])P.[dR](T)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[s
			P].[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR]
			(T)
#63	1	GÅGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[TNA](T)P.[d
			R](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#64	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[T
			NA](T)P.[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#65	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[TNA](G)P.[dR](C)[sP].[dR](C)[P].[d
			R](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#66	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[TNA]([5meC])P.[dR](C)
			[sP].[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR]
			(T)
#67	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[TNA]([5meC])
			P.[dR](A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR]
			(T)
#68	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[TNA](A)P.[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#69	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[dR](A)[sP].[TNA](A)P.[LR]([5meC])[sP].[LR](T)
#70	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].
			[dR](A)[sP].[LR](A)[sP].[TNA]([5meC])P. [LR](T)
CONTROL	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
#2		TCA	[P].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)[sP].[MOE]([5meC])[sP].[MOE](A)
#71	2	GCCAGGCTGGTTATGAC	[TNA](G)[sP].[TNA]([5meC])[sP].[TNA]([5meC])
		TCA	[sP].[TNA](A)P.[TNA](G)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#72	2	GCCAGGCTGGTTATGAC	[TNA](G)[sP].[TNA]([5meC])[sP].[TNA]([5meC])
		TCA	P.[TNA](A)P.[TNA](G)[sP].[dR](G)[sP].[dR](C)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].
			[MOE](A)[P].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#73	2	GCCAGGCTGGTTATGAC	[TNA](G)[sP].[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
		TCA	(A)P.[TNA](G)[sP].[dR](G)[sP].[dR](C)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#74	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
		TCA	(A)P.[TNA](G)[sP].[dR](G)[sP].[dR](C)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#75	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
		TCA	(A)P.[TNA](G)P.[TNA](G)[sP].[dR](C)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#76	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
		TCA	(A)P.[TNA](G)P.[TNA](G)P.[TNA]([5meC])[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			[MOE]([5meC])[sP].[MOE](A)
#77	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
		TCA	(A)P.[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA]
			(T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#78	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
		TCA	(A)P.[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA]
			(T)P.[TNA](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			[5meC])[sP].[MOE](A)
#79	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
		TCA	(A)P.[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA]
			(T)P.[TNA](G)P.[TNA](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#80	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#81	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[dR](C)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[SP].[MOE](A)[sP].[MOE]([5meC])[P].[MOE](T)[s
			P].[MOE]([5meC])[sP].[MOE](A)
#82	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)
			[sP].[dR](G)[sP].[MOE](A)[sP].[MOE]([5meC])
			[sP][MOE])(T)[sP].[MOE]([5meC])[sP].[MOE](A)
#83	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])P.[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)[sP].[MOE]([5meC])[sP].[MOE](A)
#84	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](T)[sP].[dR](G)[sP].[dR](G)[sP].[d
			R](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC]}[sP].[MOE]
			(T)[sP].[MOE]([5meC]}[sP].[MOE](A)
#85	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](T)P.[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#86	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](T)P.[TNA](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)[sP].[MOE]([5meC])[sP][MOE](A)
#87	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP][MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](T)P.[TNA](G)P.[dR](G)[P].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC][sP].[MOE](A)
#88	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](T)P.[TNA](G)P.[TNA](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP][MOE](A)
#89	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP][MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](T)P.[TNA](G)P.[TNA](G)P.[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#90	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](T)P.[TNA](G)P.[TNA](G)P.[TNA]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP][MOE](A)
#91	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[MOE]([5meC])[sP].[MOE]([5meC])[s
			P].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
		TCA	(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sp}.[dR](A)[sP.][dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])P.[TNA](A)
#92	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[MOE]([5meC])[sP].[MOE]([5meC])[s
		TCA	P].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			P.[TNA]([5meC])P.[TNA](A)
#93	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[MOE]([5meC])[sP].
		TCA	[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR](C)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[s
			P].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)P.
			[TNA]([5meC])P.[TNA](A)
#94	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[MOE]([5meC])[sP].
		TCA	[MOE](A)[sP].[MOE](G)P.[TNA](G)[sP].[dR](C)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](G)[sp].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].
			[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)P.[TN
			A]([5meC])P.[TNA](A)
#95	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[MOE]([5meC])[sP].
		TCA	[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]([5meC])
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)P.
			[TNA]([5meC])P.[TNA](A)
#96	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[MOE]([5meC])[sP].
		TCA	[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]([5meC])
			P.[TNA](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[R](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)P.
			[TNA]([5meC])P.[TNA](A)
#97	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[MOE]([5meC])[sP].
		TCA	[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]([5meC])
			P.[TNA](T)P.[TNA](G)[sP].[dR](G)[sP].[dR](T)[s
			P].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)P.
			[TNA]([5meC])P.[TNA](A)
#98	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[MOE]([5meC])[sP].
		TCA	[MOE](A)[P].[MOE](G)P.[TNA](G)P.[TNA]([5meC])
			P.[TNA](T)P.[TNA](G)P.[TNA](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].
			[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)P.[TN
			A]([5meC])P.[TNA](A)
#99	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])P.[TN
		TCA	A](A)P.[TNA](G)[sP].[dR](G)[sP].[dR](C)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[TN
			A](A)P.[TNA]([5meC])P.[TNA](T)P.[TNA]([5meC])
			P.[TNA](A)
#100	2	GCCAGGCTGGTTATGAC	[TNA](G)[sP].[TNA]([5meC])P.[TNA]([5meC])P.
		TCA	[TNA](A)P.[TNA](G)[sP].[dR](G)[sP].[dR](C)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sp].
			[TNA](A)P.[TNA]([5meC])P.[TNA](T)P.[TNA]([5meC])
			[sP].[TNA](A)
#101	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[TNA](A)P.[TNA]([5meC])P.[TNA](T)P.[T
			NA]([5meC])P.[TNA](A)
#102	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sp].[MOE](G)[sP].[dR](G)[sp].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP],[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sp].[dR](T)[sP].[TN
			A](G)P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)P.[TN
			A]([5meC])P.[TNA](A)
#103	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[TNA](T)P.[TNA]
			(G)P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)P.[TNA]
			([5meC])P.[TNA](A)
#104	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[TNA](A)P.[TNA](T)P.[TNA]
			(G)P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)P.[TNA]
			([5meC])P.[TNA](A)
#105	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)[sP].[TNA]([5meC])P.[TNA](A)
#106	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP][MOE]([5meC])
		TCA	[sP].[MOE](A)[P].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[P].[MOE]
			(T)P.[TNA]([5meC])P.[TNA](A)
#107	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[MOE]([5meC])[sP].[MOE]([5meC])[s
		TCA	P].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[SP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meCI)[sP].[MOE](T)
			[sP].[MOE]([5meC])P.[TNA](A)
#108	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])P.[TNA]([5meC])P.
		TCA	[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR](C)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[s
			P].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])P.[TNA](T)P.[MO
			E]([5meC])[sP].[MOE](A)
#109	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])P.[TNA]([5meC])P.
		TCA	[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR](C)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[s
			P].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)P.[TNA]([5meC])P.[MOE](T)[sP].[MO
			E]([5meC])[sP].[MOE](A)
#110	2	GCCAGGCTGGTTATGAC	[MOE](G)[P].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[dR](C)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)P.[TNA](G)P.
			[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#111	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])P.[TNA]([5meC])P.
		TCA	[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR](C)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[s
			P].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)P.[TNA]([5meC])P.[TNA](T)P.[MOE]
			([5meC])[sP].[MOE](A)
#112	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])P.[TNA]([5meC])P.
		TCA	[TNA](A)P.[MOE](G)[sP].[dR](G)[sP].[dR](C)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].
			[dRI(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].
			[MOE](A)P.[TNA]([5meC])P.[TNA](T)P.[MOE]
			([5meC])[sP].[MOE](A)
#113	2	GCCAGGCTGGTTATGAC	[MOE](G)P.[TNA]([5meC])P.[MOE]([5meC])[sP].
		TCA	[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR](C)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[s
			P].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC]}[sP].[MOE](A)
#114	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])P.[TNA]([5meC])P.
		TCA	[MOE](A)[P].[MOE](G)[sP].[dR](G)[sP].[dR](C)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](G)[P].[dR](T)[s
			P].[dR](T)[sP].[dR](A)[sP].[R](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP] [MOE](T)[sP].
			[MOE]([5meC)[sP].[MOE](A)
#115	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	P.[TNA](A)P.[MOE](G)[sP].[dR](G)[SP].[dR](C)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[s
			P].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#116	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)P.[TNA](G)P.[dR](G)[sP].[dR](C)[s
			P].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[s
			P].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[P].[MOE](A)
#117	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[dR](C)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[MOE](A)[sP].[MOE]([SmeC])[sP].[MOE](T)[s
			P].[MOE]([5meC])[sP].[MOE](A)
#118	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)P.[TNA]
			([5meC])P.[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[d
			R](G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)[sP].[MOE]([5meC])[sP].[MOE](A)
#119	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[R](G)[sP][dR]
			(C)P.[TNA](T)P.[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#120	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[P].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)P.[TNA](G)P.[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#121	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)P.[TNA](G)P.[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[s
			P].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#122	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)P.[TNA]
			(T)P.[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[s
			P].[MOE]([5meC])[sP].[MOE](A)
#123	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)P.[TNA](T)P.[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[s
			P].[MOE]([5meC])[sP].[MOE](A)
#124	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
			[sP].[MOE](A)[sp].[MOE](G)[sp].[dR](G0[sp].[dR]
		TCA	(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)P.[TNA](A)P.[dR](T)[sP].[dR](G)
			[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[s
			P].[MOE]([5meC])[sP].[MOE](A)
#125	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)P.[TNA](T)P.[dR](G)
			[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[s
			P].[MOE]([5meC])[sP].[MOE](A)
#126	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)P.[TNA]
			(G)P.[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[s
			P].[MOE]([5meC])[sP].[MOE](A)
#127	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)P.[TNA](A)P.[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#128	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[P].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)P.[TNA]([5meC])P.[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#129	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[P].[dR](G)[sP].[dR]
			(T)[P].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])P.[TNA](T)P.
			[MOE]([5meC])[sP].[MOE](A)
#130	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)P.[TNA]([5meC])P.[MOE](A)
#131	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)[sP].[MOE]([5meC])P.[TNA](A)
#132	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[MOE]([5meC])[sP].[MOE]([5meC])[s
		TCA	P].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[P].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#133	2	GCCAGGCTGGTTATGAC	[MOE](G)P.[TNA]([5meC])[sP].[MOE]([5meC])[s
		TCA	P].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#134	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])P.[TNA]([5meC])[s
		TCA	P].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[P].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#135	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	P.[TNA](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#136	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)P.[TNA](G)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#137	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#138	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)P.[TNA]
			([5meC])[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].
			[MOE](T)[sP].[MOE]([5meC])[sP].[MOE](A)
#139	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)P.[TNA](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#140	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)P.[TNA](G)[sP].[dR](G)[P].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#141	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[P].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)P.[TNA](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#142	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)P.[TNA]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#143	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[P].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)P.[TNA](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#144	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)P.[TNA](A)[P].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[P].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#145	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)P.[TNA](T)[sP].[dR]
			(G)[sP].[MOE](A)[P].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#146	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)P.[TNA]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#147	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)P.[TNA](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#148	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[P].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)P.[TNA]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#149	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])P.[TNA](T)
			[sP].[MOE]([5meC])[sP].[MOE](A)
#150	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)P.[TNA]([5meC])[sP].[MOE](A)
CONTROL	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
#3		TTC	[MOE](A)[sP].[MOE](T)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE]([5meC])
#151	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[P].[MOE]([5meC])[sP].
		TTC	[MOE](A)[P].[MOE](T)P.[TNA](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[P].[MOE](G)[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE]([5meC]
#152	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC]) [sP].
		TTC	[MOE[(A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[P].[MOE](T)
			[sP].[MOE]([SmeC])
#153	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)P.[TN
			A](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[d
			R](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)
			[sP].[MOE]([5meC])
#154	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)P.[TN
			A](G)P.[TNA]([5meC])[sP].[dR](A)[sP].[dR](G)[s
			P].[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[s
			P].[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE]([5meC])
#155	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)P.[TN
			A](G)P.[TNA]([5meC])P.[TNA](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE]([5meC])
#156	3	GGCATATGCAGATAATG	[TNA](G)[sP].[TNA](G)[sP].[TNA]([5meC])[sP].[T
		TTC	NA](A)P.[TNA](T)[sP].[dR](A)[sP].[dR](T)[sP].[d
			R](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[d
			R](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[SP].[MOE](T)[SP].[MOE](T)
			[sP].[MOE]([5meC])
#157	3	GGCATATGCAGATAATG	[TNA](G)[sP].[TNA](G)[sP].[TNA]([5meC])P.[TNA]
		TTC	(A)P.[TNA](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[sP].
			[MOE]([5meC])
#158	3	GGCATATGCAGATAATG	[TNA](G)[sP].[TNA](G)P.[TNA]([5meC])P.[TNA]
		TTC	(A)P.[TNA](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](T)
			[sP].[MOE](G)[P].[MOE](T)[sP][MOE](T)[sP].
			[MOE]([5meC])
#159	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](T)[s
			P].[MOE](G)[SP].[MOE](T)[sP].[MOE](T)[SP].[MOE]
			([5meC])
#160	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)P.[TNA](A)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C([sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](T)[sP].
			[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[P].[MOE]
			([5meC])
#161	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)P.[TNA](A)P.[TNA](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](T)[sP].
			[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			([5meC])
#162	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA](G)[sP].[d
			R](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].[d
			R](T)[P].[dR](A)[sP].[dR](A)[sP].[MOE](T)[sP].
			[MOE](G)[sP].[MOE](T)[SP].[MOE](T)[sP].[MOE]
			([5meC])
#163	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA]
			([5meC])[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](A)[P].[dR](A)[sP].[MOE](T)[s
			P].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			([5meC])
#164	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](A)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](T)[sP].
			[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			([5meC])
#165	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)P.[TNA](A)P.[TNA](T)P. [TNA](G)P.[TNA]
			([5meC])P.[TNA](A)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](A)[P].[dR](A)[sP].[dR](T)[sP].
			[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			([5meC])
#166	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](A)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			([5meC])
#167	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](A)P.[TNA](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)[sP].
			[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			([5meC])
#168	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](A)P.[TNA](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			([5meC])
#169	3	GGCATATGCAGATAATG	[TNA](G)P.[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]
		TTC	(A)[sP].[MOE](T)[sP].[dR](A)[P].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)
			P.[TNA]([5meC])
#170	3	GGCATATGCAGATAATG	[TNA](G)P.[MOE](G)[P].[MOE]([5meC])[sP].[MOE]
			(A)[sP].[MOE](T)[sP].[dR](A)[sP].[dR](T)[sP].
		TTC	[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)P.[TNA](T)P.
			[TNA]([5meC])
#171	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[MOE]([5meC])[sP].[MOE]
		TTC	(A)[sP].[MOE](T)[sP].[dR](A)[P].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(T)[sP].[MOE](G)[P].[MOE](T)P.[TNA](T)P.[TNA]
			([5meC])
#172	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[MOE]([5meC])[sP].[MOE]
		TTC	(A)[sP].[MOE](T)P.[TNA](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A) [sP].[MOE]
			(T)[sP].[MOE](G)[sP].[MOE](T)P.[TNA](T)P.[TNA]
			([5meC])
#173	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[MOE]([5meC])[sP].[MOE]
		TTC	(A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)[sP].[dR](G)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(T)[sP].[MOE](G)[sP].[MOE](T)P.[TNA](T)P.[TNA]
			([5meC])
#174	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[MOE]([5meC])[sP].[MOE]
		TTC	(A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)P.[TNA](G)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](T)
			[sP].[MOE](G)[P].[MOE](T)P.[TNA](T)P.[TNA]
			([5meC])
#175	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[MOE]([5meC])[sP].[MOE]
		TTC	(A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)P.[TNA](G)
			P.[TNA]([5meC])[sP].[dR](A)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(T)[sP].[MOE](G)[sP].[MOE](T)P.[TNA](T)P.[TNA]
			([5meC])
#176	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[MOE]([5meC])[sP].[MOE]
		TTC	A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)P.[TNA](G)
			P.[TNA]([5meC])P.[TNA](A)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](A)[P].[dR](A)[sP].[MOE]
			(T)[sP].[MOE](G)[sP].[MOE](T)P.[TNA](T)P.[TNA]
			([5meC])
#177	3	GGCATATGCAGATAATG	[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.
		TTC	[TNA](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](T)[SP].[dR](A)[sP].[dR](A)[sP].[TNA](T)P.
			[TNA](G)P.[TNA](T)P.[TNA](T)P.[TNA]([5meC])
#178	3	GGCATATGCAGATAATG	[TNA](G)[sP].[TNA](G)P.[TNA]([5meC])P.[TNA]
		TTC	(A)P.[TNA](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[TNA](T)
			P.[TNA](G)P.[TNA](T)P.[TNA](T)[sP].[TNA]
			([5meC])
#179	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)[SP].[R](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[TNA](T)P.[TNA](G)P.[TNA](T)P.[TNA](T)P.[TNA]
			([5meC])
#180	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[SP].[MOE](T)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[TNA](A)P.
			[TNA](T)P.[TNA](G)P.[TNA](T)P.[TNA](T)P.[TNA]
			([5meC])
#181	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)[sP].[dR](A)[P].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[TNA](A)P.[TNA](A)P.
			[TNA](T)P.[TNA](G)P.[TNA](T)P.[TNA](T)P.[TNA]
			([5meC])
#182	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[SP].[MOE](T)[SP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[TNA](T)P.[TNA](A)P.[TNA](A)P.[TNA]
			(T)P.[TNA](G)P.[TNA](T)P.[TNA](T)P.[TNA]
			([5meC])
#183	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE((A)[sP].[MOE](T)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[TNA]
			(T)P.[TNA]([5meC])
#184	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)[SP].[dR](A)[P].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)P.[TNA](T)
			P.[TNA]([5meC])
#185	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)P.[TNA]([5meC])[sP].[MOE]
		TTC	(A)[sP].[MOE](T)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)P.[TNA](T)[sP].[MOE](T)
			[sP].[MOE]([5meC])
#186	3	GGCATATGCAGATAATG	[MOE](G)[P].[MOE](G)P.[TNA]([5meC])P.[MOE]
			(A)[sP].[MOE](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
		TTC	(G)[sP].[dR](C)[P].[dR](A)[P].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(T)[sP].[MOE](G)P.[TNA](T)P.[MOE](T)[sP].[MOE]
			([5meC])
#187	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)P.[TNA]([5meC])P.[MOE]
		TTC	(A)[sP].[MOE](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(T)[sP].[TNA](G)P.[TNA](T)P.[MOE](T)[sP].[MOE]
			([5meC])
#188	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)P.[TNA]([5meC])P.[MOE]
		TTC	(A)[sP].[MOE](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(T)P.[TNA](G)P.[TNA](T)P.[MOE](T)[sP].[MOE]
			([5meC])
#189	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)P.[TNA]([5meC])P.[TNA]
		TTC	(A)P.[MOE](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(T)P.[TNA](G)P.[TNA](T)P.[MOE](T)[sP].[MOE]
			([5meC])
#190	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)P.[TNA](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)
			[sP].[MOE]([5meC])
#191	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[P].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)P.[dR](T)[sP].[d
			R](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[d
			R](A)[sP].[dR](T)[sP].[dR](A)P.[TNA](A)P.[MOE]
			(T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[sP].
			[MOE]([5meC])
CONTROL	4	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[sP].[MOE](G)[sP].[MOE]
#4		TTG	(A)[sP].[MOE](G)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE](G)
#192	4	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[sP].[MOE](G)[sP].[MOE]
		TTG	(A)[sP].[MOE](G)P.[TNA](T)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE](G)
#193	4	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[sP].[MOE](G)[sP].[MOE]
		TTG	(A)[sP].[MOE](G)P.[TNA](T)P.[TNA](T)[sP].[dR]
			(A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE](G)
#194	4	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[sP].[MOE](G)[sP].[MOE]
		TTG	(A)[sP].[MOE](G)P.[TNA](T)P.[TNA](T)P.[TNA](A)
			[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)
			[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)
			[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE](T)
			[sP].[MOE](G)
#195	4	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[SP].[MOE](G)[sP].[MOE]
		TTG	(A)[sP].[MOE](G)P.[TNA](T)P.[TNA](T)P.[TNA](A)
			P.[TNA]([5meC])[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE](G)
#196	4	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[sP].[MOE](G)[sP].[MOE]
		TTG	(A)[sP].[MOE](G)P.[TNA](T)P.[TNA](T)P.[TNA](A)
			P.[TNA]([5meC])P.[TNA](T)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[P].[MOE]
			(T)[sP].[MOE](G)
#197	4	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[P].[MOE](G)[sP].[MOE]
		TTG	(A)[sP].[MOE](G)P.[TNA](T)P.[TNA](T)P.[TNA](A)
			P.[TNA]([5meC])P.[TNA](T)P.[TNA](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE](G)
#198	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[MOE](G)[sP].[MOE](A)[sP].
		TTG	[MOE](G)[sP],[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)
			[sP].[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA]
			(G)
#199	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[MOE](G)[sP].[MOE](A)[sP].
		TTG	[MOE](G)P.[TNA](T)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA]
			(G)
#200	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[MOE](G)[sP].[MOE](A)[sP].
		TTG	[MOE](G)P.[TNA](T)P.[TNA](T)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA]
			(G)
#201	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[MOE](G)[sP].[MOE](A)[sP].
		TTG	[MOE](G)P.[TNA](T)P.[TNA](T)P.[TNA](A)[sP].[d
			R](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].[d
			R](C)[SP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA]
			(G)
#202	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[MOE](G)[sP].[MOE](A)[sP].
		TTG	[MOE](G)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA]
			([5meC])[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[s
			P].[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA]
			(G)
#203	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[MOE](G)[sP].[MOE](A)[sP].
		TTG	[MOE](G)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA]
			([5meC])P.[TNA](T)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[s
			P].[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA]
			(G)
#204	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)
			[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].
			[TNA(A)P.[TNA]([5meC)P.[TNA](T)P.[TNA](T)
			P.[TNA](G)
#205	4	GGGAGTTACTTGCCAAC	[TNA](G)[sP].[TNA](G)P.[TNA](G)P.[TNA](A)P.
		TTG	[TNA](G)[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[TNA](A)P.
			[TNA]([5meC])P.[TNA](T)P.[TNA](T)[sP].[TNA](G)
#206	4	GGGAGTTACTTGCCAAC	[TNA](G)[sP].[TNA](G)[sP].[TNA](G)[sP].[TNA](A)
		TTG	P.[TNA](G)[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)
			[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)
			[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)
			[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE](T)
			[sP].[MOE](G)
#207	4	GGGAGTTACTTGCCAAC	[TNA](G)[sP].[TNA](G)[sP].[TNA](G)P.[TNA](A)P.
		TTG	[TNA](G)[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)[sP].[MOE](T)[sP].
			[MOE](G)
#208	4	GGGAGTTACTTGCCAAC	[TNA](G)[sP].[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)[sP].[MOE](T)[sP].
			[MOE](G)
#209	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR]
			(C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			(G)
#210	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)P.[TNA](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[sP].[MOE]
			([5meC])[P].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			(G)
#211	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)P.[TNA](T)P.[TNA](T)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].[dR](C)
			[P].[dR](C)[sP].[dR](A)[sP].[MOE](A)[sP].[MOE]
			([5meC])[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			(G)
#212	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)P.[TNA](T)P.[TNA](T)P.[TNA](A)[P] [dR](C)[s
			P].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[sP].[MOE]
			([5meC])[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE](G)
#213	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA]([5meC])
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].[dR]
			(C)[sP].[dR](C)[P].[dR](A)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)[sP].[MOE](T)[sP].
			[MOE](G)
#214	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA]([5meC])
			P.[TNA](T)[sP].[dR](T)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](C)[sP].[dR](A)[sP].[MOE](A)[P].[MOE]
			([5meC])[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
			(G)
#215	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA]([5meC])
			]P.[TNA](T)[sP].[dR](T)[sP].[dR](G)[sP].[R](C)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			([5meC])[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE](G)
#216	4	GGGAGTTACTTGCCAAC	[TNA](G)P.[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA]
		TTG	(G)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA]([5meC])
			P.[TNA](T)[sP].[dR](T)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](A)[sP].[dR](C)
			[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE](G)
#217	4	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[P].[MOE](G)[sP].[MOE]
			(A)[sP].[MOE](G)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
		TTG	(A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[TNA]
			(A)P.[TNA]([5meC])P.[TNA](T)P.[TNA](T)P.[TNA]
			(G)
#218	4	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[sP].[MOE](G)[sP].[MOE]
		TTG	(A)[sP].[MOE](G)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[P].[dR]
			(G)[sP].[dR](C)[sP].[TNA]([5meC])P.[TNA](A)P.
			[TNA](A)P.[TNA]([5meC])P.[TNA](T)P.[TNA](T)P.
			[TNA](G)
CONTROL	5	TGCCTTTAGGATTCTAG	[MOE](T)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
#5		ACA	[MOE]([5meC])[sP].[MOE](T)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR]
			(T)[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#219	5	TGCCTTTAGGATTCTAG	[MOE](T)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		ACA	[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)
			[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#220	5	TGCCTTTAGGATTCTAG	[MOE](T)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		ACA	[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA](T)
			[sP].[dR](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)
			[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#221	5	TGCCTTTAGGATTCTAG	[MOE](T)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		ACA	[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA](T)
			P.[TNA](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)
			[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#222	5	TGCCTTTAGGATTCTAG	[MOE](T)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		ACA	[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA](T)
			P.[TNA](A)P.[TNA](G)[sP].[dR](G)[sP].[dR](A)[s
			P].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)
			[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE](A)[sP].
			[MOE]([5meC])[sP].[MOE](A)
#223	5	TGCCTTTAGGATTCTAG	[MOE](T)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		ACA	[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA](T)
			P.[TNA](A)P.[TNA](G)P.[TNA](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].
			[MOE](A)[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#224	5	TGCCTTTAGGATTCTAG	[MOE](T)[sP].[MOE](G)[P].[MOE]([5meC])[sP].
		ACA	[MOE]([5meC])[sP].[MOE](T)P.[TNA](T)P.[TNA](T)
			P.[TNA](A)P.[TNA](G)P.[TNA](G)P.[TNA](A)[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].
			[MOE](A)[sP].[MOE](G)[sP].[MOE](A)[P].[MOE]
			([5meC])[sP].[MOE](A)
#225	5	TGCCTTTAGGATTCTAG	[TNA](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]([5m
		ACA	eC])P.[TNA](T)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[TNA]
			(A)P.[TNA](G)P.[TNA](A)P.[TNA]([5meC])P.[TNA]
			(A)
#226	5	TGCCTTTAGGATTCTAG	[TNA](T)[sP].[TNA](G)P.[TNA]([5meC])P.[TNA]
		ACA	([5meC])P.[TNA](T)[sP].[dR](T)[sP].[dR](T)[sP].
			[dR](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR].(T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].
			[TNA](A)P.[TNA](G)P.[TNA](A)P.[TNA]([5meC])[sP].
			[TNA](A)
#227	5	TGCCTTTAGGATTCTAG	[TNA](T)[sP].[TNA](G)[sP].[TNA]([5meC])[sP].
		ACA	[TNA]([5meC])P.[TNA](T)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)
			[s].[MOE](A)[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#228	5	TGCCTTTAGGATTCTAG	[TNA](T)[sP].[TNA](G)[sP].[TNA]([5meC])P.[TNA]
		ACA	([5meC])P.[TNA](T)[sP].[dR](T)[sP].[dR](T)[sP].
			[dR](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].
			[MOE](A)[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#229	5	TGCCTTTAGGATTCTAG	[TNA](T)[sP].[TNA](G)P.[TNA]([5meC])P.[TNA]
		ACA	([5meC])P.[TNA](T)[sP].[dR](T)[sP].[dR](T)[sP].[d
			R](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)[sP].[d
			R](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].
			[MOE](A)[sP].[MOE](G)[SP].[MOE](A)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#230	5	TGCCTTTAGGATTCTAG	[TNA](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])
		ACA	P.[TNA](T)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[MOE]
			(A)[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE]
			([5meC])[sP].[MOE](A)
#231	5	TGCCTTTAGGATTCTAG	[TNA](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])
		ACA	P.[TNA](T)P.[TNA](T)[sP].[dR](T)[P].[dR](A)
			[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[MOE]
			(A)[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE]([5meC])
			[sP].[MOE](A)
#232	5	TGCCTTTAGGATTCTAG	[TNA](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]([5m
		ACA	eC])P.[TNA](T)P.[TNA](T)P.[TNA](T)[sP].[dR](A)
			[sP].[dR](G)[sP].[dR](G)[sP].[dR](A)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[MOE](A)
			[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE]([5meC])
			[sP].[MOE](A)
#233	5	TGCCTTTAGGATTCTAG	[TNA](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]([5m
		ACA	eC])P.[TNA](T)P.[TNA](T)P.[TNA](T)P.[TNA](A)[sP].
			[dR](G)[sP].[dR](G)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[MOE](A)
			[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE]([5meC])
			[sP].[MOE](A)
#234	5	TGCCTTTAGGATTCTAG	[TNA](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]([5m
		ACA	eC])P.[TNA](T)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.
			[TNA](G)[sP].[dR](G)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[MOE](A)[sP].
			[MOE](G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].
			[MOE](A)
#235	5	TGCCTTTAGGATTCTAG	[TNA](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]([5m
		ACA	eC])P.[TNA](T)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.
			[TNA](G)P.[TNA](G)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[MOE](A)[sP].
			[MOE](G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].
			[MOE](A)
#236	5	TGCCTTTAGGATTCTAG	[TNA](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]([5m
		ACA	eC])P.[TNA](T)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.
			[TNA](G)P.[TNA](G)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[dR](A)[sP].
			[MOE](G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP]
			[MOE](A)
#237	5	TGCCTTTAGGATTCTAG	[TNA](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]([5m
		ACA	eC])P.[TNA](T)P.[TNA](T)P.[TNA](T)P.[TNA](A)P.
			[TNA](G)P.[TNA](G)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].
			[MOE](A)
#238	5	TGCCTTTAGGATTCTAG	[MOE](T)[sP].[MOE](G)[sP].[MOE] ([5meC])[sP].
		ACA	[MOE]([5meC])[sP].[MOE](T)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR]
			(T)[sP].[TNA](A)P.[TNA](G)P.[TNA](A)P.[TNA]([5
			meC])P.[TNA](A)
#239	5	TGCCTTTAGGATTCTAG	[MOE](T)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		ACA	[MOE]([5meC])[sP].[MOE](T)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[TNA]
			(T)P.[TNA](A)P.[TNA](G)P.[TNA](A)P.[TNA]([5m
			eC])P.[TNA](A)
#240	5	TGCCTTTAGGATTCTAG	[MOE](T)[P].[MOE](G)[sP].[MOE]([5meC])[sP].
		ACA	[MOE]([5meC])[sP].[MOE](T)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](T)[sP].[TNA]([5meC])P.
			[TNA](T)P.[TNA](A)P.[TNA](G)P.[TNA](A)P.[TNA]
			([5meC])P.[TNA](A)
CONTROL	6	CCAGGCTGGTTATGACT	[MOE]([5meC])[sP].[MOE]([5meC])[sP].[MOE](A)
#6		CAG	[sP].[MOE](G)[sP].[MOE](G)[sP].[dR](C)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			([5meC])[sP].[MOE](A)[sP].[MOE](G)
#241	6	CCAGGCTGGTTATGACT	[TNA]([5meC])[sP].[TNA]([5meC])[sP].[TNA](A)
		CAG	[sP].[TNA](G)P.[TNA](G)[sP].[dR](C)[sP].[dR](T)
			[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)
			[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]([5m
			eC])[sP].[MOE](A)[sP].[MOE](G)
#242	6	CCAGGCTGGTTATGACT	[TNA]([5meC])[sP].[TNA]([5meC])[P].[TNA](A)P.
		CAG	[TNA](G)P.[TNA](G)[sP].[dR](C)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR](T)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]([5meC])
			[sP].[MOE](A)[sP].[MOE](G)
#243	6	CCAGGCTGGTTATGACT	[TNA]([5meC])[sP].[TNA]([5meC])P.[TNA](A)P.
		CAG	[TNA](G)P.[TNA](G)[sP].[dR](C)[sP].[dR](T)[sP].[d
			R](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR](T)[sP].[d
			R](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]([5meC])[sP].
			[MOE](A)[sP].[MOE](G)
#244	6	CCAGGCTGGTTATGACT	[TNA]([5meC])P.[TNA]([5meC])P.[TNA](A)P.[TNA]
		CAG	(G)P.[TNA](G)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](G)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].[MOE]
			([5meC])[sP].[MOE](T)[sP].[MOE]([5meC])[sP].
			[MOE](A)[sP].[MOE](G)
#245	6	CCAGGCTGGTTATGACT	[TNA]([5meC])P.[TNA]([5meC])P.[TNA](A)P.[TNA]
		CAG	(G)P.[TNA](G)P.[TNA]([5meC])[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR](T)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].
			MOE]([5meC])[sP].[MOE](T)[sP].[MOE]([5meC])
			[sP].[MOE](A)[sP].[MOE](G)
#246	6	CCAGGCTGGTTATGACT	[TNA]([5meC])P.[TNA]([5meC])P.[TNA](A)P.[TNA]
		CAG	(G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](T)[sP].[d
			R](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR](T)[sP].[d
			R](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]([5meC])
			[sP].[MOE](A)[sP].[MOE](G)
#247	6	CCAGGCTGGTTATGACT	[TNA]([5meC])P.[TNA]([5meC])P.[TNA](A)P.[TNA]
		CAG	(G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](T)P.[TNA]
			(G)[sP].[dR](G)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].
			[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]([5meC])
			[sP].[MOE](A)[sP].[MOE](G)
#248	6	CCAGGCTGGTTATGACT	[TNA]([5meC])P.[TNA]([5meC])P.[TNA](A)P.[TNA]
		CAG	(G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](T)P.[TNA]
			(G)P.[TNA)(G)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].[MOE]
			([5meC])[sP].[MOE](T)[sP].[MOE]([5meC])[sP].
			[MOE](A)[sP].[MOE](G)
CONTROL	7	TTATCAATTCACCAAGG	[MOE](T)[sP].[MOE](T)[SP].[MOE](A)[sP].[MOE]
#7		AGC	(T)[sP].[MOE]([5meC])[sP].[dR](A)[sP].[dR](A)[s
			P].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](G)[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE]
			(G)[sP].[MOE]([5meC])
#249	7	TTATCAATTCACCAAGG	[TNA](T)[sP].[TNA](T)[sP].[TNA](A)[SP].[TNA](T)
		AGC	P.[TNA]([5meC])[sP].[dR](A)[sP].[dR](A)[sP].[dR]
			(T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR]
			(C)[sP].[dR](C)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(G)[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE](G)
			[sP].[MOE]([5meC])
#250	7	TTATCAATTCACCAAGG	[TNA](T)[P].[TNA](T)[sP].[TNA](A)P.[TNA](T)P.
		AGC	[TNA]([5meC])[sP].[dR](A)[P].[dR](A)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(G)[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE](G)[sP].
			[MOE]([5meC])
#251	7	TTATCAATTCACCAAGG	[TNA](T)[sP].[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA]
		AGC	([5meC])[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[s
			P].[dR](C)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](G)
			[sP].[MOE](G)[sP].[MOE](A)[sP].[MOE](G)[sP].
			[MOE]([5meC])
#252	7	TTATCAATTCACCAAGG	[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA]
		AGC	([5meC])[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](G)[sP].
			[MOE](G)[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE]
			([5meC])
#253	7	TTATCAATTCACCAAGG	[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA]
		AGC	[5meC])P.[TNA](A)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](G)[sP].
			[MOE](G)[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE]
			([5meC])
#254	7	TTATCAATTCACCAAGG	[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA]
		AGC	([5meC])P.[TNA](A)P.[TNA](A)[sP].[dR](T)[sP].[d
			R](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].[d
			R](C)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](G)[sP].
			[MOE](G)[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE]
			([5meC])
#255	7	TTATCAATTCACCAAGG	[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA]
		AGC	([5meC])P.[TNA](A)P.[TNA](A)P.[TNA](T)[P].[dR]
			(T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].[dR]
			(C)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](G)[sP].
			[MOE](G)[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE]
			([5meC])
#256	7	TTATCAATTCACCAAGG	[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA]
		AGC	([5meC])P.[TNA](A)P.[TNA](A)P.[TNA](T)P.[TNA]
			(T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].[dR]
			(C)[sP].[dR](A)[sP].[dR](A)[sP].[MOE](G)[sP].
			[MOE](G)[sP].[MOE](A)[sP].[MOE](G)[sP].[MOE]
			([5meC])
#257	7	TTATCAATTCACCAAGG	[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA]
		AGC	([5meC])P.[TNA](A)P.[TNA](A)P.[TNA](T)P.[TNA]
			(T)P.[TNA]([5meC])
			[sP].[dR](A)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)
			[sP].[dR](A)[sP].[MOE](G)[sP].[MOE](G)[P].[MOE]
			(A)[sP].[MOE](G)[sP].[MOE]([5meC])
CONTROL	8	ATGGAGGTATGACATAT	[MOE](A)[sP].[MOE](T)[sP].[MOE](G)[sP].[MOE]
#8		AAT	(G)[sP].[MOE](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](C)[sP].[dR](A)[sP].[dR](T)[sP].[MOE]
			(A)[sP].[MOE](T)[sP].[MOE](A)[sP].[MOE](A)[sP].
			[MOE](T)
#258	8	ATGGAGGTATGACATAT	[TNA](A)[sP].[TNA](T)[sP].[TNA](G)[sP].[TNA](G)
		ÅAT	P.[TNA](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](T)[sP].[MOE](A)
			[sP].[MOE](T)[P].[MOE](A)[sP].[MOE](A)[sP].
			[MOE](T)
#259	8	ATGGAGGTATGACATAT	[TNA](A)[sP].[TNA](T)[sP].[TNA](G)P.[TNA](G)P.
		AAT	[TNA](A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](T)[sP].[MOE](A)[sP].
			[MOE](T)[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE]
			(T)
#260	8	ATGGAGGTATGACATAT	[TNA](A)[sP].[TNA](T)P.[TNA](G)P.[TNA](G)P.[TNA]
		AAT	(A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](T)[sP].[MOE](A)[sP].
			[MOE](T)[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE]
			(T)
#261	8	ATGGAGGTATGACATAT	[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA](G)P.[TNA]
		AAT	(A)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].[dR]
			(C)[sP].[dR](A)[sP].[dR](T)[sP].[MOE](A)[sP].[MOE]
			(T)[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE](T)
#262	8	ATGGAGGTATGACATAT	[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA](G)P.[TNA]
		AAT	(A)P.[TNA](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].[dR]
			(C)[sP].[dR](A)[sP].[dR](T)[sP].[MOE](A)[SP].[MOE]
			(T)[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE](T)
#263	8	ATGGAGGTATGACATAT	[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA](G)P.[TNA]
		AAT	(A)P.[TNA](G)P.[TNA](G)[sP].[dR](T)[sP].[dR](A)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](A)[sP].[dR](T)[sP].[MOE](A)[sP].[MOE]
			(T)[sP].[MOE](A)[sP].[MOE](A)[P].[MOE](T)
#264	8	ATGGAGGTATGACATAT	[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA](G)P.[TNA]
		AAT	(A)P.[TNA](G)P.[TNA](G)P.[TNA](T)[sP].[dR](A)
			[SP].[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](A)[sP].[dR](T)[sP].[MOE](A)[sP].[MOE]
			(T)[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE](T)
#265	8	ATGGAGGTATGACATAT	[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA](G)P.[TNA]
		AAT	(A)P.[TNA](G)P.[TNA](G)P.[TNA](T)P.[TNA](A)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](A)[sP].[dR](C)[sP].
			[dR](A)[sP].[dR](T)[sP].[MOE](A)[sP].[MOE](T)
			[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE](T)
#266	8	ATGGAGGTATGACATAT	[TNA](A)P.[TNA](T)P.[TNA](G)P.[TNA](G)P.[TNA]
		AAT	(A)P.[TNA](G)P.[TNA](G)P.[TNA](T)P.[TNA](A)P.
			[TNA](T)[sP].[dR](G)[sP].[dR](A)[sP].[dR](C)[sP].
			[dR](A)[sP].[dR](T)[sP].[MOE](A)[sP].[MOE](T)
			[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE](T)
#267	9	GTTATGCTTATTCCCCA	[TNA](G)[sP].[TNA](T)P.[TNA](T)P.[TNA](A)P.[TNA]
		ATG	(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](T)[sP].[dR]
			(C)[sP].[dR](C)[sP].[dR](C)[sP].[TNA]([5meC])P.
			[TNA](A)P.[TNA](A)P.[TNA](T)[sP].[TNA](G)
#268	10	CTCAGCCTTTATCACTC	[TNA]([5meC])[sP].[TNA](T)P.[TNA]([5meC])P.[TNA]
		AGC	(A)P.[TNA](G)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](T)[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR]
			(T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].[TNA]
			(T)P.[TNA]([5meC])P.[TNA](A)P.[TNA](G)[sP].
			TNA]([5meC]
#269	11	AACAAATTTCCTTAGTT	[TNA](A)[sP].[TNA](A)P.[TNA]([5meC])P.[TNA](A)
		GGC	P.[TNA](A)[sP].[dR](A)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](C)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](G)[sP].[TNA](T)
			P.[TNA](T)P.[TNA](G)P.[TNA](G)[sP].[TNA]([5meC])
#270	12	TACACCAGTOCTTTTAG	[TNA](T)[sP].[TNA](A)P.[TNA]([5meC])P.[TNA](A)
		TAG	P.[TNA]([5meC])[sP].[dR](C)[sP].[dR](A)[sP].[d
			R](G)[sP].[dR](T)[sP].[dR](C)[sP].[dR](C)[sP].[d
			R](T)[sP].[dR](T)[sP].[dR](T)[sP].[dR](T)[sP].[TNA]
			(A)P.[TNA](G)P.[TNA](T)P.[TNA](A)[sP].[TNA]
			(G)
#271	13	ATGCTCATCACTTTATG	[TNA](A)[sP].[TNA](T)P.[TNA](G)P.[TNA]([5meC])
		AAG	P.[TNA](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](T)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[TNA](T)
			P.[TNA](G)P.[TNA](A)P.[TNA](A)[sP].[TNA](G)
#272	14	AAAAGGCTTAGCGCCCA	[TNA](A)[sP].[TNA](A)P.[TNA](A)P.[TNA](A)P.[TNA]
		CCT	(G)[sP].[dR](G)[sP].[dR](C)[sP].[dR](T)[sP][d
			R](T)[sP].[dR](A)[sP].[dR](G)[sP].[dR]([5meC])
			[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[TNA]
			([5meC])P.[TNA](A)P.[TNA]([5meC])P.[TNA]([5meC])
			[sP].[TNA](T)
#273	15	AAGAGAACCACACACTA	[TNA](A)[SP].[TNA](A)P.[TNA](G)P.[TNA](A)P.[TNA]
		CCA	(G)[sP].[dR](A)[sP].[dR](A)[sP].[dR](C)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].[TNA](T)P.
			[TNA](A)P.[TNA]([5meC])P.[TNA]([5meC])[sP].
			[TNA](A)
#274	16	CATCTCAACCTCCGTCA	[TNA]([5meC])[sP].[TNA](A)P.[TNA](T)P.[TNA]
		TGT	([5meC])P.[TNA](T)[sP].[dR](C)[sP].[dR](A)[sP].
			[dR](A)[sP].[dR](C)[P].[dR](C)[sP].[dR](T)[sP].
			[dR](C)[sP].[dR]([5meC])[sP].[dR](G)[sP].[dR](T)
			[sP].[TNA]([5meC])P.[TNA](A)P.[TNA](T)P.[TNA]
			(G)[sP].[TNA](T)
#275	17	AAGGTCTCATACACTCA	[TNA](A)[SP].[TNA](A)P.[TNA](G)P.[TNA](G)P.
		CTA	[TNA](T)[sP].[dR](C)[sP].[dR](T)[sP].[dR](C)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](C)[sP].
			[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[TNA]([5meC])
			P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)[sP].[TNA]
			(A)
#276	18	GTACTATCCCATCACTG	[TNA](G)[sP].[TNA](T)P.[TNA](A)P.[TNA]([5meC])
		AAG	P.[TNA](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](C)
			[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[dR](T)[s
			P].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].[TNA](T)
			P.[TNA](G)P.[TNA](A)P.[TNA](A)[sP].[TNA](G)
#277	19	GACCCCTGACTTTCTGG	[TNA](G)[sP].[TNA](A)P.[TNA]([5meC])P.[TNA]
		AAA	([5meC])P.[TNA]([5meC])[sP].[dR](C)[sP].[dR](T)
			[sP].[dR](G)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[P].[dR](T)[s
			P].[TNA](G)P.[TNA](G)P.[TNA](A)P.[TNA](A)[sP].
			[TNA](A)
#278	20	CAGCGGTACACTCCTTC	[TNA]([5meC])[sP].[TNA](A)P.[TNA](G)P.[TNA]
		TCT	([5meC])P.[TNA](G)[sP].[dR](G)[sP].[dR](T)[sP].[d
			R](A)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].[d
			R](T)[sP].[dR](C)[sP].[dR](C)[sP].[dR](T)[sP].
			[TNA](T)P.[TNA]([5meC])P.[TNA](T)P.[TNA]([5meC])
			[sP].[TNA](T)
#279	21	GCCAATATTTGCCCCTC	[TNA](G)[sP].[TNA]([5meC])P.[TNA]([5meC])P.
		CCC	[TNA](A)P.[TNA](A)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].
			[dR](C)[SP].[dR](C)[sP].[dR](C)[sP].[dR](C)[sP].
			[TNA](T)P.[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
			([5meC])[sP].[TNA]([5meC])
#280	22	TGCATTTACTTGCCAAC	[TNA](T)[sP].[TNA](G)P.[TNA]([5meC])P.[TNA](A)
		AGA	P.[TNA](T)[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)
			[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)
			[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[TNA](A)
			P.[TNA]([5meC])P.[TNA](A)P.[TNA](G)[sP].[TNA]
			(A)
#281	23	AAAGAGTAACTACCAGC	[TNA](A)[sP].[TNA](A)P.[TNA](A)P.[TNA](G)P.[TNA]
		CAT	(A)[sP].[dR](G)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[TNA](G)P.
			[TNA]([5meC])P.[TNA]([5meC])P.[TNA](A)[sP].
			[TNA](T)
#282	24	AACAGGTCATCTATTCA	[TNA](A)[sP].[TNA](A)P.[TNA]([5meC])P.[TNA](A)
		CAA	P.[TNA](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR](C)
			[sP].[dR](A)[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)
			[sP].[dR](A)[sP].[dR](T)[sP].[dR](T)[sP].[TNA]
			([5meC])P.[TNA](A)P.[TNA]([5meC])P.[TNA](A)[sP].
			[TNA](A)
#283	25	TGTACATTTTGCCCTTA	[TNA](T)[sP].[TNA](G)P.[TNA](T)P.[TNA](A)P.[TN
		GCT	A]([5meC])[sP]/[dR](A)[sp].[dR](T)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](C)[sP].[dR](C)[sP].[dR](T)[sP.[TNA](T)
			P.[TNA](A)P.[TNA](G)P.[TNA]([5meC])[sP].[TNA]
			(T)
#284	26	GCCAAGCACTCATATGC	[TNA](G)[sP].[TNA]([5meC])P.[TNA]([5meC])P.
		AAT	[TNA](A)P.[TNA](A)[sP].[dR](G)[sP].[dR](C)[sP].
			[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](C)[sP].
			9dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].
			[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.[TNA](A)[sP].
			[TNA](T)
#285	27	GTTATGCTTATTCCCCA	[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE]
		ATG	(A)[sP].[MOE](T)P.[TNA](G)P.[TNA]([5meC])P.[TNA]
			(T)P.[TNA](T)P.[TNA](A)P.[TNA](T)[sP].[dR](T)
			[sP].[dR](C)[sP].[dR](C)[sP].[dR](C)[sP].[MOE]
			([5meC])[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE](T)
			[sP].[MOE](G)
#286	28	GTGTAATTACCTTTTACT	[MOE](G)[sP].[MOE](T)[SP].[MOE](G)[sP].[MOE]
		CT	(T)[sP].[MOE](A)P.[TNA](A)P.[TNA](T)P.[TNA](T)
			P.[TNA](A)P.[TNA]([5meC])P.[TNA]([5meC])[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](T)[sP].[dR](T)[sP].
			[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[sP].[MOE](T)
#287	29	CCTTCAGAGATTCAATG	[MOE]([5meC])[sP].[MOE]([5meC])[sP].[MOE](T)
		CTA	[sP].[MOE](T)[sP].[MOE]([5meC])P.[TNA](A)P.[T
			NA](G)P.[TNA](A)P.[TNA](G)P.[TNA](A)P.[TNA](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](A)
			[sP].[MOE](T)[sP].[MOE](G)[sP].[MOE]([5meC])
			[sP].[MOE](T)[sP].[MOE](A)
#288	30	GCCAATATTTGCCCCTC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		CCC	[sP].[MOE](A)[sP].[MOE](A)P.[TNA](T)P.[TNA](A)
			P.[TNA](T)P.[TNA](T)P.[TNA](T)P.[TNA](G)[sP].
			[dR](C)[sP].[dR](C)[sP].[dR](C)[sP].[dR](C)[sP].
			[MOE](T)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
			[sP].[MOE]([5meC])[sP].[MOE]([5meC])
#289	31	TGCTAAACAAATTTCCT	[MOE](T)[P].[MOE](G)[sP].[MOE]([5meC])[sP].
		TAG	[MOE](T)[sP].[MOE](A)P.[TNA](A)P.[TNA](A)P.[TN
			A]([5meC])P.[TNA](A)P.[TNA](A)P.[TNA](A)[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].
			[MOE]([5meC])[sP].[MOE](T)[sP].[MOE](T)[sP].
			[MOE](A)[sP].[MOE](G)
#290	32	AACAAATTTOCTTAGTT	[MOE](A)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].
		GGC	[MOE](A)[sP].[MOE](A)P.[TNA](A)P.[TNA](T)P.[TN
			A](T)P.[TNA](T)P.[TNA]([5meC])P.[TNA]([5meC])
			[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](G)
			[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE](G)[sP].
			[MOE](G)[sP].[MOE]([5meC])
#291	33	TCAGTGCTATTTTATOC	[MOE](T)[sP].[MOE]([5meC])[sP].[MOE](A)[P].
		AAT	[MOE](G)[P].[MOE](T)P.[TNA](G)P.[TNA]([5meC])
			P.[TNA](T)P.[TNA](A)P.[TNA](T)P.[TNA](T)[P].
			[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].
			[MOE]([5meC])[sP].[MOE]([5meC])[sP].[MOE](A)
			[sP].[MOE](A)[sP].[MOE](T)
#292	34	TGTACATTTTGCCCTTA	[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[SP].[MOE]
		GCT	(A)[sP].[MOE]([5meC])P.[TNA](A)P.[TNA](T)P.[TN
			A](T)P.[TNA](T)P.[TNA](T)P.[TNA](G)[sP].[dR](C)
			[sP].[dR](C)[sP].[dR](C)[sP].[dR](T)[sP].[MOE]
			(T)[sP].[MOE](A)[sP].[MOE](G)[P].[MOE]([5meC])
			[sP].[MOE](T)
#293	35	TTGCTGAAATTGTCTCA	[MOE](T)[sP].[MOE](T)[sP].[MOE](G)[sP].[MOE]
		ATT	([5meC])[sP].[MOE](T)P.[TNA](G)P.[TNA](A)P.[TN
			A](A)P.[TNA](A)P.[TNA](T)P.[TNA](T)[sP].[dR](G)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](T)[sP].[MOE]
			([5meC])[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE](T)
			[sP].[MOE](T)
#294	36	TTGAAGCATACCTTAAC	[MOE](T)[sP].[MOE](T)[sP].[MOE](G)[sP].[MOE]
		ATC	(A)[sP].[MOE](A)P.[TNA](G)P.[TNA]([5meC])P.[TN
			A](A)P.[TNA](T)P.[TNA](A)P.[TNA]([5meC])[sP].
			[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].
			[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](A)[sP].
			[MOE](T)[sP].[MOE]([5meC])
#295	37	CTCCAATACTTGTCTTA	[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]([5meC])
		GCT	[sP].[MOE]([5meC])[sP].[MOE](A)P.[TNA](A)P.[T
			NA](T)P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)P.[T
			NA](T)[sP].[dR](G)[sP].[dR](T)[sP].[dR](C)[sP].
			[dR](T)[sP].[MOE](T)[sP].[MOE](A)[sP].[MOE](G)
			[sP].[MOE]([5meC])[sP].[MOE](T)
#296	38	GCCTTAAAGTTACATTC	MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		GTT	[sP].[MOE](T)[sP].[MOE](T)P.[TNA](A)P.[TNA](A)
			P.[TNA](A)P.[TNA](G)P.[TNA](T)P.[TNA](T)[sP].
			[dR](A)[sP].[dR](C)[sP].[dR](A)[sP].[dR](T)[sP].
			MOE](T)[sP].[MOE]([5meC])[sP].[MOE](G)[sP].
			[MOE](T)[sP].[MOE](T)
#297	39	AAGGTCTCATACACTCA	[MOE](A)[sP].[MOE](A)[sP].[MOE](G)[SP].[MOE]
		CTA	G)[sP].[MOE](T)P.[TNA]([5meC])P.[TNA](T)P.[TN
			A]([5meC])P.[TNA](A)P.[TNA](T)P.[TNA](A)[sP].
			[MOE]([5meC])[sP].[MOE](A)[sP].[MOE]([5meC])
			[sP].[MOE](T)[sP].[MOE](A)
			[MOE]([5meC)][sP].[MOE](A)[sP].[MOE]([5meC)]
			[sP].[MOE](T)[sP].[MOE](A)
#298	40	GTACTATCCCATCACTG	[MOE](G)[sP].[MOE](T)[P].[MOE](A)[sP].[MOE]
		AAG	([5meC])[sP].[MOE](T)P.[TNA](A)P.[TNA](T)P.[TNA]
			([5meC])P.[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
			(A)[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].
			[dR](C)[sP].[MOE](T)[sP].[MOE](G)[sP].[MOE](A)
			[sP].[MOE](A)[sP].[MOE](G)
#299	41	ATGCAAGTTAAACTTAT	[MOE](A)[sP].[MOE](T)[sP].[MOE](G)[sP].[MOE]
		CTG	([5meC])[sP].[MOE](A)P.[TNA](A)P.[TNA](G)P.[TN
			A](T)P.[TNA](T)P.[TNA](A)P.[TNA](A)[P].[dR](A)
			[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].[MOE](A)
			[sP].[MOE](T)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE](G)
#300	42	TGCCTCTTCATTGTATTT	[MOE](T)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		CT	[MOE]([5meC])[P].[MOE](T)P.[TNA]([5meC])P.[T
			NA](T)P.[TNA](T)P.[TNA]([5meC])P.[TNA](A)P.[T
			NA](T)[sP].[dR](T)[sP].[dR](G)[sP].[dR](T)[sP].
			[dR](A)[sP].[MOE](T)[sP].[MOE](T)[sP].[MOE](T)
			[sP].[MOE]([5meC)][sP].[MOE](T)
#301	43	CATCAAGGCACTGATCA	[MOE]([5meC])[P].[MOE](A)[sP].[MOE](T)[sP].
		CTT	[MOE]([5meC])[sP].[MOE](A)P.[TNA](A)P.[TNA](G)
			P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)P.[TNA]
			([5meC])[sP].[dR](T)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](T)[sP].[MOE]([5meC)][sP].[MOE](A)
			[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE](T)
#302	44	GCCAAGCACTCATATGC	[MOE](G)[sP].[MOE]([5meC])[sP].[MOE]([5meC])
		AAT	[sP].[MOE](A)[sP].[MOE](A)P.[TNA](G)P.[TNA]
			([5meC])P.[TNA](A)P.[TNA]([5meC])P.[TNA](T)P.
			[TNA]([5meC])[sP].[dR](A)[sP].[dR](T)[sP].[dR]
			(A)[sP].[dR](T)[sP].[MOE](G)[sP].[MOE]([5meC])
			[sP].[MOE](A)[sP].[MOE](A)[sP].[MOE](T)
#303	45	GGGAGTTACTTGCCAAC	[MOE](G)[sP].[MOE](G)[sP].[MOE](G)[sP].[MOE]
		TTG	(A)[sP].[MOE](G)P.[TNA](T)P.[TNA](T)P.[TNA](A)
			P.[TNA]([5meC])P.[TNA](T)P.[TNA](T)[sP].[dR]
			(G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].[MOE]
			(A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].[MOE]
			(T)[sP].[MOE](G)
#304	46	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[dR](T)[sP].[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#305	46	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[dR](T)P.[dR](T)
			[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)
			[sP].[LR](A)[SP].[LR]([5meC])[sP].[LR](T)
#306	46	GAGTTACTTGCCAACT	[LR](G)[P].[LR](A)[SP].[LR](G)P.[dR](T)P.[dR](T)
			P.[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].
			[LR](A)[sP].[LR](5meC])[sP].[LR](T)
#307	46	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[dR](T)P.[dR](T)
			P.[dR](A)P.[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)[sP].
			[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#308	46	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[dR](T)P.[dR](T)
			P.[dR](A)P.[dR](C)P.[dR](T)[sP].[dR](T)[sP].[dR]
			(G)[sP].[dR](C)[P].[dR](C)[sP].[dR](A)[sP].[LR]
			(A)[sP].[LR]([5meC])[sP].[LR](T)
CONTROL	47	CTTTGGCAGAATGAGG	[LR]([5meC])[sP].[LR](T)[sP].[LR](T)[sP].[dR](T)
#9			[sP].[dR](G)[sP].[dR](G)[sP].[dR](C)[sP].[dR](A)
			[sP].[dR](G)[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)
			[sP].[dR](G)[sP].[LR](A)[sP].[LR](G)[sP].[LR](G)
#309	47	CTTTGGCAGAATGAGG	[LR]([5meC])[sP].[LR](T)[sP].[LR](T)P.[TNA](T)
			[sP].[dR](G)[sP].[dR](G)[sP].[dR](C)[sP].[dR](A)
			[sP].[dR](G)[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)
			[sP].[dR](G)[sP].[LR](A)[sP].[LR](G)[sP].[LR](G)
#310	47	CTTTGGCAGAATGAGG	[LR]([5meC])[sP].[LR](T)[sP].[LR](T)P.[TNA](T)P.
			[TNA](G)[sP].[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].
			[dR](G)[sP].[dR](A)[sP].[dR](A)[P].[dR](T)[sP].
			[dR](G)[sP].[LR](A)[sP].[LR](G)[sP].[LR](G)
#311	47	CTTTGGCAGAATGAGG	[LR]([5meC])[sP].[LR](T)[sP].[LR](T)P.[TNA](T)P.
			[TNA](G)P.[TNA](G)[sP].[dR](C)[sP].[dR](A)[sP].
			[dR](G)[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[LR](A)[sP].[LR](G)[sP].[LR](G)
#312	47	CTTTGGCAGAATGAGG	[LR]([5meC])[sP].[LR](T)[sP].[LR](T)P.[TNA](T)P.
			[TNA](G)P.[TNA](G)P.[TNA]([5meC])[sP].[dR](A)
			[sP].[dR](G)[sP].[dR](A)[P].[dR](A)[sP].[dR](T)
			[sP].[dR](G)[sP].[LR](A)[sP].[LR](G)[sP].[LR](G)
#313	47	CTTTGGCAGAATGAGG	[LR]([5meC])[sP].[LR](T)[sP].[LR](T)P.[TNA](T)P.
			[TNA](G)P.[TNA](G)P.[TNA]([5meC])P.[TNA](A)
			[sP].[dR](G)[sP].[dR](A)[sP].[dR](A)[sP].[dR](T)
			[sP].[dR](G)[sP].[LR](A)[sP].[LR](G)[sP].[LR](G)
CONTROL	48	GCTTTGGCAGAATGAG	[LR](G)[sP].[LR]([5meC])[sP].[LR](T)[sP].[dR](T)
#10			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].[dR](A)
			[sP].[dR](T)[sP].[LR](G)[sP].[LR](A)[sP].[LR](G)
#314	48	GCTTTGGCAGAATGAG	[LR](G)[sP].[LR]([5meC])[sP].[LR](T)P.[TNA](T)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](C)
			[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].[dR](A)
			[sP].[dR](T)[sP].[LR](G)[sP].[LR](A)[sP].[LR](G)
#315	48	GCTTTGGCAGAATGAG	[LR](G)[P].[LR]([5meC])[sP].[LR](T)P.[TNA](T)P.
			[TNA](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](C)[sP].
			[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].[dR](A)[sP].
			[dR](T)[sP].[LR](G)[sP].[LR](A)[sP].[LR](G)
#316	48	GCTTTGGCAGAATGAG	[LR](G)[sP].[LR]([5meC])[sP].[LR](T)P.[TNA](T)P.
			[TNA](T)P.[TNA](G)[sP].[dR](G)[sP].[dR](C)[sP].
			[dR](A)[sP].[dR](G)[sP].[dR](A)[P].[dR](A)[sP].
			[dR](T)[sP].[LR](G)[sP].[LR](A)[sP].[LR](G)
#317	48	GCTTTGGCAGAATGAG	[LR](G)[sP].[LR]([5meC])[sP].[LR](T)P.[TNA](T)P.
			[TNA](T)P.[TNA](G)P.[TNA](G)[sP].[dR](C)[sP].
			[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].[dR](A)[sP].
			[dR](T)[sP].[LR](G)[sP].[LR](A)[sP].[LR](G)
#318	48	GCTTTGGCAGAATGAG	[LR](G)[sP].[LR]([5meC])[sP].[LR](T)P.[TNA](T)P.
			[TNA](T)P.[TNA](G)P.[TNA](G)P.[TNA]([5meC])
			[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].[dR](A)
			[sP].[dR](T)[sP].[LR](G)[sP].[LR](A)[sP].[LR](G)
CONTROL	49	ACATGCTTTCACAGAAT	[LR](A)[sP].[LR]([5meC])[sP].[dR](A)[sP].[dR](T)
#11		T	[P].[dR](G)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](A)[sP].[dR](G)[sP].[LR](A)[P].[LR](A)
			[sP].[LR](T)[sP].[LR](T)
#319	49	ACATGCTTTCACAGAAT	[LR](A)[sP].[LR]([5meC])P.[TNA](A)[sP].[dR](T)
		T	[sP].[dR](G)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](A)[sP].[dR](G)[sP].[LR](A)[sP].[LR](A)
			[sP].[LR](T)[sP].[LR](T)
#320	49	ACATGCTTTCACAGAAT	[LR](A)[sP].[LR]([5meC])P.[TNA](A)P.[TNA](T)
		T	[sP].[dR](G)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](A)[sP].[dR](G)[sP].[LR](A)[sP].[LR](A)
			[sP].[LR](T)[sP].[LR](T)
#321	49	ACATGCTTTCACAGAAT	[LR](A)[sP].[LR]([5meC])P.[TNA](A)P.[TNA](T)P.
		T	[TNA](G)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)[sP].
			[dR](A)[sP].[dR](G)[sP].[LR](A)[sP].[LR](A)[sP].
			[LR](T)[sP].[LR](T)
#322	49	ACATGCTTTCACAGAAT	[LR](A)[sP].[LR]([5meC])P.[TNA](A)P.[TNA](T)P.
		T	[TNA](G)P.[TNA]([5meC])[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](A)[sP].[dR](G)[sP].[LR](A)[sP].[LR](A)
			[sP].[LR](T)[sP].[LR](T)
#323	49	ACATGCTTTCACAGAAT	[LR](A)[sP].[LR]([5meC])P.[TNA](A)P.[TNA](T)P.
		T	[TNA](G)P.[TNA]([5meC])P.[TNA](T)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)[sP].[dR](C)
			[sP].[dR](A)[P].[dR](G)[sP].[LR](A)[sP].[LR](A)
			[sP].[LR](T)[sP].[LR](T)
CONTROL	50	TTCATTCACAGACAGGA	[LR](T)[sP].[LR](T)[sP].[LR]([5meC])[sP].[LR](A)
#12			[sP].[dR](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[LR](G)
			[sP].[LR](A)
#324	50	TTCATTCACAGACAGGA	[LR](T)[sP].[LR](T)[sP].[LR]([5meC])[sP].[LR](A)
			P.[TNA](T)[sP].[dR](T)[sP].[dR](C)[sP].[dR](A)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[P].[LR](G)
			[sP].[LR](A)
#325	50	TTCATTCACAGACAGGA	[LR](T)[P].[LR](T)[P].[LR]([5meC])[sP].[LR](A)
			P.[TNA](T)P.[TNA](T)[sP].[dR](C)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)[sP].
			[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[LR](G)[sP].
			[LR](A)
#326	50	TTCATTCACAGACAGGA	[LR](T)[sP].[LR](T)[sP].[LR]([5meC])[sP].[LR](A)
			P.[TNA](T)P.[TNA](T)P.[TNA]([5meC])[sP].[dR](A)
			[sP].[dR](C)[P].[dR](A)[sP].[dR](G)[P].[dR](A)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[LR](G)
			[sP].[LR](A)
#327	50	TTCATTCACAGACAGGA	[LR](T)[sP].[LR](T)[sP].[LR]([5meC])[sP].[LR](A)
			P.[TNA](T)P.[TNA](T)P.[TNA]([5meC])P.[TNA](A)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR](A)
			[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[LR](G)
			[sP].[LR](A)
#328	50	TTCATTCACAGACAGGA	[LR](T)[sP].[LR](T)[sP].[LR]([5meC])[sP].[LR](A)
			P.[TNA](T)P.[TNA](T)P.[TNA]([5meC])P.[TNA](A)
			P.[TNA]([5meC])[sP].[dR](A)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[LR]
			(G)[sP].[LR](A)
#329	1	GAGTTACTTGCCAACT	[LR](G)[P].[LR](A)[sP].[LR](G)P.[TNA](T)P.[TNA]
			(T)P.[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR](A)
			[sP].[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#330	1	GAGTTACTTGCCAACT	[LR](G)[sP].[LR](A)[sP].[LR](G)P.[TNA](T)P.[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR](T)
			[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)P.[TNA](A)P.
			[LR](A)[sP].[LR]([5meC])[sP].[LR](T)
#331	1	GAGTTACTTGCCAACT	[LR](G)[PS2].[LR](A)[sP].[LR](G)P.[TNA](T)P.[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[PS2].[LR](T)
#332	1	GAGTTACTTGCCAACT	[LR](G)[PS2].[LR](A)[sP].[LR](G)[sP].[dR](T)P.[T
			NA](T)P.[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[P].[dR](C)[P].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[PS2].[LR](T)
#333	1	GAGTTACTTGCCAACT	[LR](G)[PS2].[LR](A)[sP].[LR](G)P.[TNA](T)P.[TN
			A](T)P.[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)[sP].[dR]
			(A)[sP].[LR](A)[sP].[LR]([5meC])[PS2].[LR](T)
#334	1	GAGTTACTTGCCAACT	[LR](G)[PS2].[LR](A)[sP].[LR](G)P.[TNA](T)P.[dR]
			(T)[sP].[dR](A)[sP].[dR](C)[sP].[dR](T)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](C)[sP].[dR](C)P.[TNA](A)
			P.[LR](A)[sP].[LR]([5meC])[PS2].[LR](T)
#335	2	GCCAGGCTGGTTATGAC	[MOE](G)[PS2].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[dR](C)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR[(G)
			[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)
			[sP].[MOE]([5meC])[PS2].[MOE](A)
#336	2	GCCAGGCTGGTTATGAC	[MOE](G)[PS2].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)[sP].[dR](G)P.[TNA]
			([5meC])P.[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)[sP].[MOE]([5meC])[PS2].[MOE](A)
#337	2	GCCAGGCTGGTTATGAC	[MOE](G)[PS2].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[TNA]
			([5meC])P.[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].
			[dR](T)[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].
			[dR](G)[sP].[MOE](A)[sP].[MOE]([5meC])[sP].[MOE]
			(T)[sP].[MOE]([5meC])[PS2].[MOE](A)
#338	2	GCCAGGCTGGTTATGAC	[MOE](G)[PS2].[MOE]([5meC])[sP].[MOE]([5meC])
		TCA	[sP].[MOE](A)[sP].[MOE](G)P.[TNA](G)P.[dR](C)
			[sP].[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)
			[sP].[dR](T)[sP].[dR](A)[sP].[dR](T)P.[TNA](G)P.
			[MOE](A)[sP].[MOE]([5meC])[sP].[MOE](T)[sP].
			[MOE]([5meC])[PS2].[MOE](A)
#339	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)P.[dR](T)[sP].[d
			R](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[d
			R](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)
			[sP].[MOE]([5meC])
#340	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)[sP].[dR](A)P.[TNA](T)P.[d
			R](G)[P].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[d
			R](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)
			[sP].[MOE]([5meC])
#341	3	GGCATATGCAGATAATG	[MOE](G)[sP].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)P.[dR]
			(G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR]
			(A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].[MOE]
			(T)[sP].[MOE](G)[P].[MOE](T)[sP].[MOE](T)[sP].
			[MOE]([5meC])
#342	3	GGCATATGCAGATAATG	[MOE](G)[PS2].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)P.[dR](T)[sP].
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)
			[PS2].[MOE]([5meC])
#343	3	GGCATATGCAGATAATG	[MOE](G)[PS2].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)[sP].[dR](A)P.[TNA](T)P.
			[dR](G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)
			[PS2].[MOE]([5meC])
#344	3	GGCATATGCAGATAATG	[MOE](G)[PS2].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)P.[TNA](T)P.[dR]
			(G)[sP].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].[dR]
			(A)[P].[dR](T)[sP].[dR](A)[sP].[dR](A)[sP].
			[MOE](T)[sP].[MOE](G)[sP].[ MOE](T)[sP].[MOE](T)
			[PS2].[MOE]([5meC])
#345	3	GGCATATGCAGATAATG	[MOE](G)[PS2].[MOE](G)[sP].[MOE]([5meC])[sP].
		TTC	[MOE](A)[sP].[MOE](T)P.[TNA](A)P.[dR](T)[sP].
			[dR](G)[P].[dR](C)[sP].[dR](A)[sP].[dR](G)[sP].
			[dR](A)[sP].[dR](T)[sP].[dR](A)P.[TNA](A)P.[MOE]
			(T)[sP].[MOE](G)[sP].[MOE](T)[sP].[MOE](T)[PS2].
			[MOE]([5meC])
#346	2	GCCAGGCTGGTTATGAC	[MOE](G)[sP].[MOE]([5meC])P.[MOE]([5meC])P.
		TCA	[MOE](A)P.[MOE](G)[sP].[dR](G)[sP].[dR](C)[sP].
			[dR](T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].
			[dR](T)[sP].[dR](A)[sP].[dR](T)[sP].[dR](G)[sP].
			[MOE](A)P.[MOE]([5meC])P.[MOE](T)P.[MOE]
			([5meC])[sP].[MOE](A)
#347	2	GCCAGGCTGGTTATGAC	[TNA](G)P.[TNA]([5meC])P.[TNA]([5meC])P.[TNA]
		TCA	(A)P.[TNA](G)[sP].[dR](G)[sP].[dR](C)[sP].[dR]
			(T)[sP].[dR](G)[sP].[dR](G)[sP].[dR](T)[sP].[dR]
			(T)[P].[dR](A)[P].[dR](T)[sP].[dR](G)[sP].[TNA]
			(A)P.[TNA]([5meC])P.[TNA](T)P.[TNA]([5meC])
			P.[TNA](A)

Example 2: In Vitro Efficacy of Oligonucleotides Targeting MALAT1 RNA in A549 Cells at Two Different Concentrations (5 and 25 MM)

A549 cell line was purchased from ATCC and maintained as recommended by the supplier in a humidified incubator at 37° C. with 5% CO2. For assays, 3000 cells/well were seeded in a 96 multi well plate in full culture media. Cells were incubated for 24 hours before addition of oligonucleotides dissolved in PBS for final concentrations as indicated. 3 days after addition of oligonucleotides, the cells were harvested. RNA was extracted using the RNeasy 96 RNA Purification kit (Qiagen) according to the manufacturer's instructions and eluated in 50 μL of water. The RNA was subsequently diluted 10 times with DNase/RNase free water and heated to 90° C. for one minute.

For gene expression analysis, One Step RT-qPCR was performed using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quantabio) in a duplex setup. The following TaqMan primer assays were used for qRT-PCR: MALAT1, Hs00273907_s1 [FAM-MGB], CERS2, Hs01017465_m1 [FAM-MGB], CERS5, Hs00332291_m1 [FAM-MGB], CERS6, Hs00826756_m1 [FAM-MGB] and endogenous control GAPDH, Hs99999905_m1 [VIC-MGB-PL]. All primer sets were purchased from Thermo Fisher Scientific. The relative RNA expression level of MALAT1, CERS2, CERS5, or CERS6, also referred to as the knock-down (KD) value, was calculated as percent of control (PBS-treated cells).

Determination of KD values were performed using GraphPad Prism.

The results are shown in Tables 7-10. Values separated by “/” indicate the individual results when the compound or control was tested in more than one test vial.

TABLE 7
Knock-down (KD) values targeting MALAT1

		KD		KD
CMP ID		(25 μM)	SD	(5 μM)	SD
NO	Sequence	(%)	(%)	(%)	(%)
CONTROL #1	GAGttacttgccaAmCT	14/7	0/1	30/19	2/1
#1	GAGoTtacttgccaAmCT	10	0	24	1
#2	GAGoToTacttgccaAmCT	14	0	28	2
#3	GAGoToToActtgccaAmCT	13	0	31	0
#4	GAGoToToAomCttgccaAmCT	18	0	39	1
#5	GAGoToToAomCoTtgccaAmCT	35	4	72	2
#6	GAoGttacttgccaAmCT	4	1	14	2
#7	GoAoGttacttgccaAmCT	6	0	21	4
#8	GoAoGoTtacttgccaAmCT	10	2	28	5
#9	GoAoGoToTacttgccaAmCT	11	0	27	4
#10	GoAoGoToToActtgccaAmCT	8	1	31	6
#11	GoAoGoToToAomCttgccaAmCT	16	1	41	1
#12	GoAoGoToToAomCoTtgccaAmCT	32	2	63	0
#13	GoAoGoToToActtgccaamCT	4	0	13	2
#14	GoAoGoToToActtgccaacT	3	0	9	1
#15	GoAoGoToToAomCttgccaamCT	4	0	20	0
#16	GoAoGoToToAomCttgccaacT	2	0	12	1
#17	GoAoGoToToAomCoTtgccaamCT	5	0	18	0
#18	GoAoGoToToAomCoTtgccaacT	3	0	13	3
#19	GoAGttacttgccaAmCoT	10	0	20	0
#20	GoAGttacttgccaAomCoT	8	1	21	1
#21	GoAoGttacttgccaAomCoT	57	4	75	2
#22	GAoGttacttgccaoAomCT	21	3	36	1
#23	GAoGottacttgccaoAomCT	17	1	43	6
#24	GAoGottacttgccaAomCT	4	0	18	3
#25	GAoGottacttgccaAomCT	5	0	18	0
#26	GAoGottacttgccaAomCoT	7	0	30	1
#27	GoAGttacttgccaAomCT	7	1	30	0
#28	GoAoGttacttgccaAomCT	38	0	62	0
#29	GoAoGttacttgccaAmCT	11	2	29	2
#30	GAoGottacttgccaAmCT	7	1	18	1
#31	GAGoTotacttgccaAmCT	7	1	19	0
#32	GAGtoToacttgccaAmCT	10	1	27	0
#33	GAGttoAocttgccaAmCT	18	1	36	0
#34	GAGttaomCottgccaAmCT	19	0	43	0
#35	GAGttacoTotgccaAmCT	23	4	40	1
#36	GAGttactoTogccaAmCT	26	3	45	1
#37	GAGttacttoGoccaAmCT	27	2	61	3
#38	GAGttacttgomCocaAmCT	51	5	78	2
#39	GAGttacttgcomCoaAmCT	44	3	66	4
#40	GAGttacttgccoAoAmCT	25	2	49	3
#41	GAGttacttgccaoAomCT	9	1	19	1
#42	GAGttacttgccaAomCoT	3	0	6	0
#43	GAGttacttgccaAmCoT	4	0	16	2
#44	GoAGttacttgccaAmCT	8	0	29	2
#45	GAoGttacttgccaAmCT	4	0	14	1
#46	GAGoTtacttgccaAmCT	9	0	31	0
#47	GAGtoTacttgccaAmCT	16	0	37	2
#48	GAGttoActtgccaAmCT	10	0	27	2
#49	GAGttaomCttgccaAmCT	13	1	39	3
#50	GAGttacoTtgccaAmCT	18	0	48	1
#51	GAGttactoTgccaAmCT	18	3	42	4
#52	GAGttacttoGccaAmCT	30	0	49	0
#53	GAGttacttgomCcaAmCT	40	2	64	4
#54	GAGttacttgcomCaAmCT	25	0	39	3
#55	GAGttacttgccoAAmCT	16	2	30	1
#56	GAGttacttgccaoAmCT	6	1	17	1
#57	GAoGttacttgccaAmCT	8	0	20	1
#58	GAGottacttgccaAmCT	4	1	14	2
#59	GAGTotacttgccaAmCT	4	2	7	0
#60	GAGtToacttgccaAmCT	14	0	33	3
#61	GAGttAocttgccaAmCT	17	1	33	3
#62	GAGttamCottgccaAmCT	16	4	43	5
#63	GAGttacTotgccaAmCT	23	0	60	8
#64	GAGttactTogccaAmCT	27	2	66	3
#65	GAGttacttGoccaAmCT	29	0	67	3
#66	GAGttacttgmCocaAmCT	64	4	87	20
#67	GAGttacttgcmCoaAmCT	55	3	76	2
#68	GAGttacttgccAoAmCT	15	0	53	4
#69	GAGttacttgccaAomCT	7	0	24	7
#70	GAGttacttgccaAmCoT	3	0	6	0
CONTROL #2	GmCmCAGgctggttatgAmCTmCA	12/8	1/1	17/14	0/0
#71	GmCmCAoGgctggttatgAmCTmCA	5	0	10	1
#72	GmCmCoAoGgctggttatgAmCTmCA	4	0	8	2
#73	GmComCoAoGgctggttatgAmCTmCA	4	0	7	1
#74	GomComCoAoGgctggttatgAmCTmCA	3	0	6	1
#75	GomComCoAoGoGctggttatgAmCTmCA	5	0	11	0
#76	GomComCoAoGoGomCtggttatgAmCTmCA	6	1	16	1
#77	GomComCoAoGoGomCoTggttatgAmCTmCA	8	1	18	1
#78	GomComCoAoGoGomCoToGgttatgAmCTmCA	14	2	23	2
#79	GomComCoAoGoGomCoToGoGttatgAmCTmCA	16	1	26	0
#80	GmCmCAGoGctggttatgAmCTmCA	8	1	14	2
#81	GmCmCAGoGoctggttatgAmCTmCA	8	2	15	1
#82	GmCmCAGoGomCtggttatgAmCTmCA	9	0	13	2
#83	GmCmCAGoGomCotggttatgAmCTmCA	7	1	13	2
#84	GmCmCAGoGomCoTggttatgAmCTmCA	14	3	18	3
#85	GmCmCAGoGomCoToggttatgAmCTmCA	13	1	25	0
#86	GmCmCAGoGomCoToGgttatgAmCTmCA	11	0	19	1
#87	GmCmCAGoGomCoToGogttatgAmCTmCA	17	3	39	1
#88	GmCmCAGoGomCoToGoGttatgAmCTmCA	16	1	24	1
#89	GmCmCAGoGomCoToGoGottatgAmCTmCA	18	2	38	5
#90	GmCmCAGoGomCoToGoGoTtatgAmCTmCA	28	4	46	1
#91	GomCmCAGgctggttatgAmCTmCoA	11	1	29	0
#92	GomCmCAGgctggttatgAmCTomCoA	17	1	28	1
#93	GomComCAGgctggttatgAmCTomCoA	13	0	26	0
#101	GmCmCAGgctggttatgAomCoTomCoA	10	1	14	1
#102	GmCmCAGgctggttatGoAomCoTomCoA	20	3	30	1
#103	GmCmCAGgctggttaToGoAomCoTomCoA	34	3	45	3
#104	GmCmCAGgctggttAoToGoAomCoTomCoA	33	4	37	2
#105	GmCmCAGgctggttatgAmCTmCoA	10	0	14	1
#106	GmCmCAGgctggttatgAmCTomCoA	11	1	18	1
#107	GomCmCAGgctggttatgAmCTomCoA	9	1	16	0
#108	GmComCoAGgctggttatgAmCoTomCA	16	1	27	6
#109	GmComCoAGgctggttatgAomCoTmCA	13	1	21	0
#110	GmCmCAGoGoctggttatoGoAmCTmCA	21	1	31	1
#111	GmComCoAGgctggttatgAomCoTomCA	22	1	40	3
#112	GmComCoAoGgctggttatgAomCoTomCA	27	2	52	5
#113	GomComCAGgctggttatgAmCTmCA	8	1	12	1
#114	GmComCoAGgctggttatgAmCTmCA	6	1	13	0
#115	GmCmCoAoGgctggttatgAmCTmCA	7	2	14	2
#116	GmCmCAoGogctggttatgAmCTmCA	7	0	12	0
#117	GmCmCAGoGoctggttatgAmCTmCA	9	0	12	2
#118	GmCmCAGgomCotggttatgAmCTmCA	9	1	13	1
#119	GmCmCAGgcoToggttatgAmCTmCA	10	1	16	0
#120	GmCmCAGgctoGogttatgAmCTmCA	8	0	11	1
#121	GmCmCAGgctgoGottatgAmCTmCA	13	1	24	0
#122	GmCmCAGgctggoTotatgAmCTmCA	9	1	16	1
#123	GmCmCAGgctggtoToatgAmCTmCA	12	0	21	0
#124	GmCmCAGgctggttoAotgAmCTmCA	21	3	25	2
#125	GmCmCAGgctggttaoTogAmCTmCA	29	2	25	4
#126	GmCmCAGgctggttatoGoAmCTmCA	20	0	23	1
#127	GmCmCAGgctggttatgoAomCTmCA	20	1	25	3
#128	GmCmCAGgctggttatgAomCoTmCA	12	1	16	0
#129	GmCmCAGgctggttatgAmCoTomCA	13	1	15	0
#130	GmCmCAGgctggttatgAmCTomCoA	12	2	22	4
#131	GmCmCAGgctggttatgAmCTmCoA	10	1	16	3
#132	GomCmCAGgctggttatgAmCTmCA	7	1	11	1
#133	GomCmCAGgctggttatgAmCTmCA	6	0	13	1
#134	GmComCAGgctggttatgAmCTmCA	6	0	11	2
#135	GmCmCoAGgctggttatgAmCTmCA	6	0	10	1
#136	GmCmCAoGgctggttatgAmCTmCA	6	0	9	1
#137	GmCmCAGoGctggttatgAmCTmCA	13	1	19	3
#138	GmCmCAGgomCtggttatgAmCTmCA	12	2	14	1
#139	GmCmCAGgcoTggttatgAmCTmCA	17	3	20	1
#140	GmCmCAGgctoGgttatgAmCTmCA	13	1	15	1
#141	GmCmCAGgctgoGttatgAmCTmCA	12	1	12	1
#142	GmCmCAGgctggoTtatgAmCTmCA	14	1	14	1
#143	GmCmCAGgctggtoTatgAmCTmCA	21	1	21	1
#144	GmCmCAGgctggttoAtgAmCTmCA	17	0	18	1
#145	GmCmCAGgctggttaoTgAmCTmCA	30	4	34	6
#146	GmCmCAGgctggttatoGAmCTmCA	31	5	28	0
#147	GmCmCAGgctggttatgoAmCTmCA	16	1	29	5
#148	GmCmCAGgctggttatgAomCTmCA	17	4	21	1
#149	GmCmCAGgctggttatgAmCoTmCA	18	6	26	2
#150	GmCmCAGgctggttatgAmCTomCA	12	3	17	2
CONTROL #3	GGmCATatgcagataaTGTTmC	9	2	12	2
#151	GGmCAToAtgcagataaTGTTmC	12	1	17	6
#152	GGmCAToAoTgcagataaTGTTmC	12	1	15	2
#153	GGmCAToAoToGcagataaTGTTmC	18	1	24	5
#154	GGmCAToAoToGomCagataaTGTTmC	22	0	31	5
#155	GGmCAToAoToGomCoAgataaTGTTmC	26	3	36	6
#156	GGmCAoTatgcagataaTGTTmC	5	1	9	2
#157	GGmCoAoTatgcagataaTGTTmC	3	0	8	0
#158	GGomCoAoTatgcagataaTGTTmC	4	0	8	0
#159	GoGomCoAoTatgcagataaTGTTmC	3	0	8	2
#160	GoGomCoAoToAtgcagataaTGTTmC	5	0	13	1
#161	GoGomCoAoToAoTgcagataaTGTTmC	6	0	11	0
#162	GoGomCoAoToAoToGcagataaTGTTmC	11	0	23	0
#163	GoGomCoAoToAoToGomCagataaTGTTmC	13	4	19	0
#164	GoGomCoAoToAoToGomCoAgataaTGTTmC	19	3	29	0
#165	GoGomCoAoToAoToGomCoAgataatGTTmC	5	1	10	0
#166	GoGomCoAoToAoToGomCoAgataatgTTmC	4	1	9	1
#167	GoGomCoAoToAoToGomCoAoGataatGTTmC	18	7	30	3
#168	GoGomCoAoToAoToGomCoAoGataatgTTmC	12	1	27	2
#169	GoGmCATatgcagataaTGTTomC	5	0	10	0
#170	GoGmCATatgcagataaTGToTomC	6	0	6	0
#171	GoGomCATatgcagataaTGToTomC	5	0	7	1
#179	GGmCATatgcagataaToGoToTomC	8	2	13	0
#180	GGmCATatgcagataAoToGoToTomC	10	1	17	1
#181	GGmCATatgcagatAoAoToGoToTomC	12	0	28	1
#182	GGmCATatgcagaToAoAoToGoToTomC	18	0	46	0
CONTROL #4	GGGAGttacttgccaAmCTTG	5/4/6	0/0/2	8/11/12	1/1/1
#192	GGGAGoTtacttgccaAmCTTG	7	2	14	4
#193	GGGAGoToTacttgccaAmCTTG	7	1	16	1
#194	GGGAGoToToActtgccaAmCTTG	7	1	17	1
#195	GGGAGoToToAomCttgccaAmCTTG	7	1	19	5
#196	GGGAGoToToAomCoTtgccaAmCTTG	11	2	26	1
#197	GGGAGoToToAomCoToTgccaAmCTTG	13	1	24	1
#204	GoGoGoAoGttacttgccaAomCoToToG	4	0	8	1
#205	GGoGoAoGttacttgccaAomCoToTG	4	1	8	1
#206	GGGAoGttacttgccaAmCTTG	4	0	5	0
#207	GGGoAoGttacttgccaAmCTTG	4	0	7	0
#208	GGoGoAoGttacttgccaAmCTTG	3	0	9	0
#209	GoGoGoAoGttacttgccaAmCTTG	4	0	7	1
#210	GoGoGoAoGoTtacttgccaAmCTTG	3	0	7	1
#211	GoGoGoAoGoToTacttgccaAmCTTG	2	0	4	0
#212	GoGoGoAoGoToToActtgccaAmCTTG	3	1	5	0
#213	GoGoGoAoGoToToAomCttgccaAmCTTG	6	0	10	0
#214	GoGoGoAoGoToToAomCoTttgccaAmCTTG	6/5	1/0	17/11	1/1
#215	GoGoGoAoGoToToAomCoTttgccaamCTTG	3	0	7	0
#216	GoGoGoAoGoToToAomCoTttgccaacTTG	1	0	3	0
#217	GGGAGttacttgccaAomCoToToG	5	0	16	3
#218	GGGAGttacttgcmCoAoAomCoToToG	27	3	66	3
CONTROL #5	TGmCmCTttaggattctAGAmCA	12/3	1/0	23/8	0/0
#219	TGmCmCToTtaggattctAGAmCA	16	2	32	4
#220	TGmCmCToToTaggattctAGAmCA	9	1	14	2
#221	TGmCmCToToToAggattctAGAmCA	13	2	24	3
#222	TGmCmCToToToAoGgattctAGAmCA	14	2	29	4
#223	TGmCmCToToToAoGoGattctAGAmCA	38	2	59	2
#224	TGmCmCToToToAoGoGoAttctAGAmCA	29	2	56	4
#227	TGmCmCoTttaggattctAGAmCA	23	1	43	13
#228	TGmComCoTttaggattctAGAmCA	15	1	33	2
#229	TGomComCoTttaggattctAGAmCA	13	1	29	2
#230	ToGomComCoTttaggattctAGAmCA	10	0	28	3
#231	ToGomComCoToTtaggattctAGAmCA	10	0	28	4
#232	ToGomComCoToToTaggattctAGAmCA	11	1	21	2
#233	ToGomComCoToToToAggattctAGAmCA	13	1	33	2
#234	ToGomComCoToToToAoGgattctAGAmCA	7	0	13	1
#235	ToGomComCoToToToAoGoGattctAGAmCA	17/18	1/2	24/31	1/7
#236	ToGomComCoToToToAoGoGattctaGAmCA	20	1	35	2
#237	ToGomComCoToToToAoGoGattctagAmCA	11	0	22	2
#238	TGmCmCTttaggattctAoGoAomCoA	13	3	38	8
#239	TGmCmCTttaggattcToAoGoAomCoA	34	0	46	1
#240	TGmCmCTttaggattmCoToAoGoAomCoA	41	0	52	0
CONTROL #6	mCmCAGGctggttatgamCTmCAG	4	0	10	2
#241	mCmCAGoGctggttatgamCTmCAG	3	0	6	0
#242	mCmCAoGoGctggttatgamCTmCAG	2	0	6	1
#243	mCmCoAoGoGctggttatgamCTmCAG	3	1	6	0
#244	mComCoAoGoGctggttatgamCTmCAG	6	0	11	2
#245	mComCoAoGoGomCtggttatgamCTmCAG	6	0	14	1
#246	mComCoAoGoGomCoTggttatgamCTmCAG	6	1	13	1
#247	mComCoAoGoGomCoToGgttatgamCTmCAG	8	0	16	4
#248	mComCoAoGoGomCoToGoGttatgamCTmCAG	8	1	21	0
#267	GToToAoTgcttattcccmCoAoAoTG	15	1	27	0
#268	mCTomCoAoGcctttatcacToCoAoGmC	17	0	33	3
#269	AAomCoAoAatttccttagToToGoGmC	10	0	23	1
#270	TAomCoAomCcagtccttttAoGoToAG	6	0	13	0
#271	AToGomCoTcatcactttaToGoAoAG	7	0	16	2
#272	AAoAoAoGgcttagmcgccmCoAomComCT	17	1	28	1
#273	AAoGoAoGaaccacacacToAomComCA	11	0	26	0
#274	mCAoTomCoTcaacctcmcgtmCoAoToGT	6	0	14	0
#275	AAoGoGoTctcatacactmCoAomCoTA	7	0	22	1
#276	GToAomCoTatcccatcacToGoAoAG	9	0	16	1
#277	GAomComComCctgactttctGoGoAoAA	15	1	29	1
#278	mCAoGomCoGgtacactcctTomCoToCT	3	0	6	0
#279	GmComCoAoAtatttgccccTomComComCmC	9	0	23	4
#280	TGomCoAoTttacttgccaAomCoAoGA	8	0	15	0
#281	AAoAoGoAgtaactaccaGomComCoAT	9	0	23	1
#282	AAomCoAoGgtcatctattmCoAomCoAA	5	0	13	0
#283	TGoToAomCattttgccctToAoGomCT	2	0	6	0
#284	GmComCoAoAgcactcatatGomCoAoAT	10	0	25	1
#285	GTTAToGomCoToToAoTtcccmCAATG	11	0	19	0
#286	GTGTAoAoToToAomComCttttAmCTmCT	6	1	14	1
#287	mCmCTTmCoAoGoAoGoAoTtcaaTGmCTA	11	1	20	1
#288	GmCmCAAoToAoToToToGccccTmCmCmCmC	11	1	21	1
#289	TGmCTAoAoAomCoAoAoAtttcmCTTAG	9	0	20	2
#290	AAmCAAoAoToToTomComCttagTTGGmC	10	0	16	1
#291	TmCAGToGomCoToAoToTttatmCmCAAT	4	0	8	0
#292	TGTAmCoAoToToToToGccctTAGmCT	9	1	14	1
#293	TTGmCToGoAoAoAoToTgtcTmCAATT	7	1	14	1
#294	TTGAAoGomCoAoToAomCcttaAmCATTC	10	0	19	1
#295	mCTmCmCAoAoToAomCoToTgtctTAGmCT	9	1	15	1
#296	GmCmCTToAoAoAoGoToTacatTmCGTT	10	1	21	2
#297	AAGGTomCoTomCoAoToAcactmCAmCTA	14	0	30	1
#298	GTAmCToAoTomComComCoAtcacTGAAG	12	0	24	1
#299	ATGmCAoAoGoToToAoAacttATmCTG	11	0	20	1
#300	TGmCmCTomCoToTomCoAoTtgtaTTTmCT	7	0	11	0
#301	mCATmCAoAoGoGomCoAomCtgatmCAmCTT	10	1	15	1
#302	GmCmCAAoGomCoAomCoTomCatatGmCAAT	14	1	24	1
#303	GGGAGoToToAomCoToTgccaAmCTTG	8	0	12	0

The data demonstrated that TNA (PO) was well tolerated with regards to KD in A549 cells when TNA was placed in flanks/gap.

TABLE 8
Knock-down (KD) values targeting MALAT1;
DNA PO vs TNA PO

		KD		KD
		(25		(5
		μM)	SD	μM)	SD
CMP ID NO	Sequence	(%)	(%)	(%)	(%)
CONTROL	GAGttacttgccAmCT	18	0	31	0
#1
#1	GAGoTtacttgccaAmCT	10	0	24	1
#2	GAGoToTacttgccaAmCT	14	0	28	2
#3	GAGoToToActtgccAmCT	13	0	31	0
#4		18	0	39	1
#5		35	4	72	2
#304	GAGottacttgccaAmCT	20	1	50	0
#305	GAGototacttgccaAmCT	44	1	72	1
#306	GAGototoacttgccaAmCT	57	0	78	3
#307	GAGototoaocttgccaAmCT	68	1	92	2
#308	GAGototoaocottgccaAmCT	88	1	95	2

The data demonstrated the superiority of TNA (PO) over DNA (PO) when TNA was placed in the DNA gap by virtue of the great metabolic stability of TNA (PO).

TABLE 9
Knock-down (KD) values targeting CERS2/5/6

			KD (25 μM)	SD	KD (5 μM)	SD
CMP ID NO	Target	Sequence	(%)	(%)	(%)	(%)

CONTROL #9	CERS2	mCTTtggcagaatgAGG	7	1	26	1
#390	CERS2	mCTToTggcagaatgAGG	10	0	41	4
#310	CERS2	mCTToToGgcagaatgAGG	24	0	55	1
#311	CERS2	mCTToToGoGcagaatgAGG	38	14	68	0
#312	CERS2		51	8	70	9
#313	CERS2		66	7	78	8
CONTROL #10	CERS2	GmCTttggcagaatGAG	2	0	18	0
#314	CERS2	GmCToTtggcagaatGAG	9	0	38	1
#315	CERS2	GmCToToTggcagaatGAG	6	0	32	4
#316	CERS2	GmCToToToGgcagaatGAG	12	2	36	10
#317	CERS2	GmCToToToGoGcagaatGAG	44	3	75	0
#318	CERS2		77	20	94	18
CONTROL #11	CERS5	AmCatgctttcacagAATT	21	2	42	2
#319	CERS5	AmCoAtgctttcacagAATT	23	0	56	9
#320	CERS5	AmCoAoTgctttcacagAATT	18	1	50	2
#321	CERS5	AmCoAoToGctttcacagAATT	19	3	45	3
#322	CERS5		19	1	58	5
#323	CERS5		20	1	47	4
CONTROL #12	CERS6	TTmCAttcacagacagGA	13	2	36	0
#324	CERS6	TTmCAoTtcacagacagGA	25	2	54	2
#325	CERS6	TTmCAoToTcacagacagGA	6	0	37	0
#326	CERS6		24	1	69	4
#327	CERS6		42	2	78	3
#328	CERS6		67	1	87	6

The data demonstrated that the strategy of using TNA to reduce DNA gap closed to 5′ wing could be generalized to other targets beyond MALAT1.

TABLE 10
Knock-down (KD) values of MALAT1 compounds
containing TNA(PO) and phosphorodithioate

		KD
		(25
		μM)	SD
CMP ID NO	Sequence	(%)	(%)

CONTROL	GAGttacttgccaAmCT	8	0
#1
#31	GAGoTotacttgccaAmCT	7	0
#32	GAGtoToacttgccaAmCT	12	1
#329	GAGoToToacttgccaAmCT	11	2
#330	GAGoTotacttgccoAoAmCT	36	9
#331	G*AGoToactttgccaAmC*T	5	0
#332	G*AGtoToacttgccaAmC*T	9	0
#333	G*AGoToToacttgccaAmC*T	6	1
#334	G*AGoTotacttgccoAoAmC*T	31	2
CONTROL	GmCmCAGgctggttatgAmCTmCA	7	1
#2
#81	GmCmCAGoGoctggttatgAmCTmCA	9	0
#118		8	1
#83		9	0
#110	GmCmCAGoGoctggttatoGoAmCTmCA	16	2
#335	G*mCmCAGoGoctggttatgAmCTmC*A	7	0
#336		9	0
#337		12	0
#338	G*mCmCAGoGoctggttatoGoAmCTmC*A	15	1
CONTROL	GGmCATatgcagataaTGTTmC	6	0
#3
#339	GGmCAToAotgcagataaTGTTmC	4	0
#340	GGmCATaoTogcagataaTGTTmC	7	0
#341	GGmCAToAoTogcagataaTGTTmC	8	0
#191	GGmCAToAotgcagataoAoTGTTmC	14	1
#342	G*GmCAToAotgcagataaTGTT*mC	5	0
#343	G*GmCATaoTogcagataaTGTT*mC	5	0
#344	G*GmCAToAoTogcagataaTGTT*mC	7	1
#345	G*GmCAToAotgcagataoAoTGTT*mC	15	2

The new architecture containing TNA (PO) in DNA gap and phosphorodithioate (PS2) at both termini was successfully demonstrated. Phosphorodithioate linkages greatly reduced MALAT1 expression at 25 μM in A549 cells.

Example 3: In Vitro Potency and Efficacy of Oligonucleotides Targeting MALAT1 RNA in A549 Cells at Different Concentrations for a Dose Response Curve

A549 cell lines were purchased from ATCC and maintained as recommended by the supplier in a humidified incubator at 37° C. with 5% CO2. For assays, 3500 cells/well (A549) were seeded in a 96 multi well plate in culture media. Cells were incubated for 24 hours before addition of oligonucleotides dissolved in PBS. Concentration range of oligonucleotides: highest concentration 25 μM, 1:1 dilutions in 8 steps. Three days after addition of oligonucleotides, the cells were harvested. RNA was extracted using the PureLink Pro 96 RNA Purification kit (Thermo Fisher Scientific) according to the manufacturer's instructions and eluated in 50 μL of water. The RNA was subsequently diluted 10 times with DNase/RNase free Water (Gibco) and heated to 90° C. for one minute.

For gene expression analysis, One Step RT-qPCR was performed using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quantabio) in a duplex setup. The following TaqMan primer assays were used for qPCR: MALAT1, Hs00273907_s1 (FAM-MGB) with endogenous control GAPDH. All primer sets were purchased from Thermo Fisher Scientific. The relative expression level of MALAT1 RNA is shown as percent of control (PBS-treated cells) and IC50 values have been determined using GraphPad Prism7 on data from n=2 biological replicates.

The results are shown in Table 11 below. Values separated by “/” indicate the individual results when the compound or control was tested in more than one test vial.

TABLE 11
In vitro potency results (IC50)

CMP ID NO	Sequence	IC50 (N = 2) [μM]
CONTROL #1	GAGttacttgccaAmCT	0.58/1.31
#1	GAGoTtacttgccaAmCT	1.04
#2	GAGoToTacttgccaAmCT	1.98
#3	GAGoToToActtgccaAmCT	1.36
#4		2.33
#5		4.60
#6	GAoGttacttgccaAmCT	0.37
#7	GoAoGttacttgccaAmCT	0.53
#8	GoAoGoTtacttgccaAmCT	0.94
#9	GoAoGoToTacttgccAmCT	0.78
#10	GoAoGoToToActtgccaAmCT	0.74
#11		1.49
#12		6.11
#19	GoAGttacttgccaAmCoT	0.34
#20		0.41
#21		4.46
#22	GAoGttacttgccaoAomCT	2.98
#23	GAoGottacttgccaoAomCT	3.51
#24		1.01
#25		0.61
#26		0.81
#27		0.94
#28		9.03
#29	GoAoGttacttgccaAmCT	1.6
#30	GAoGottacttgccaAmCT	0.5
#31	GAGoTotacttgccaAmCT	0.5
#32	GAGtoToacttgccaAmCT	1.1
#33	GAGttoAocttgccaAmCT	1.6
#34		2.7
#35	GAGttacoTotgccaAmCT	1.4
#36	GAGttactoTogccaAmCT	4.1
#37	GAGttacttoGoccaAmCT	3.1
#38		4.7
#39		3.2
#40	GAGttacttgccoAoAmCT	0.8
#41	GAGttacttgccaoAomCT	0.5
#42		0.1
#43	GAGttacttgccaAmCoT	0.3
CONTROL #2	GmCmCAGgctggttatgAmCTmCA	0.46/0.26/0.74
#71		0.16
#72		0.20
#73		0.18
#74		0.13
#75		0.41
#76		0.38
#77		0.52
#78		0.52
#79		0.84
#80	GmCmCAGoGctggttatgAmCTmCA	0.78
#81	GmCmCAGoGoctggttatgAmCTmCA	0.54
#82		0.53
#83		0.74
#84		0.88
#85		1.91
#86		1.07
#87		1.71
#88		2.49
#89		4.80
#90		8.51
#91	GomCmCAGgctggttatgAmCTmCoA	0.28
#92		0.26
#93		0.36/0.43
#94		1.17
#95		0.67
#96		0.89
#97		0.45
#98		0.99
#99		0.94
#100		0.35
#101		3.11
#102		3.22
#103		7.44
#104		7.20
#105		0.33
#106		0.58
#107	GomCmCAGgctggttatgAmCTomCoA	0.30
#108		1.03
#109		1.31
#110	GmCmCAGoGoctggttatoGoAmCTmCA	1.71
#111		3.10
#112		3.81
#113		0.45
#114		0.37
#115	GmCmCoAoGgctggttatgAmCTmCA	0.31
#116	GmCmCAoGogctggttatgAmCTmCA	0.49
#117	GmCmCAGoGoctggttatgAmCTmCA	0.59
#118		0.71
#119	GmCmCAGgcoToggttatgAmCTmCA	0.88
#120	GmCmCAGgctoGogttatgAmCTmCA	0.56
#121	GmCmCAGgctgoGottatgAmCTmCA	0.69
#122	GmCmCAGgctggoTotatgAmCTmCA	0.66
#123	GmCmCAGgctggtoToatgAmCTmCA	1.02
#124	GmCmCAGgctggttoAotgAmCTmCA	1.42
#125	GmCmCAGgctggttaoTogAmCTmCA	0.97
#126	GmCmCAGgctggttatoGoAmCTmCA	0.55
#127	GmCmCAGgctggttatgoAomCTmCA	0.97
#128		0.91
#129	GmCmCAGgctggttatgAmCoTomCA	0.68
#130		0.41
#131	GmCmCAGgctggttatgAmCTmCoA	0.48
CONTROL #3	GGmCATatgcagataaTGTTmC	0.21/0.17
#151	GGmCAToAtgcagataaTGTTmC	0.17
#152	GGmCAToAoTgcagataaTGTTmC	0.26
#153	GGmCAToAoToGcagataaTGTTmC	0.38
#154		0.56
#155		0.61
#156		0.13
#157		0.16
#158		0.23
#159		0.15
#160		0.11
#161		0.14
#162		0.45
#163		0.57
#164		0.57
#169		0.24
#170		0.71
#171		0.13/0.15
#172		0.26
#173		0.21
#174		0.70
#175		1.76
#176		4.33
#177		0.83
#178		0.65
#179		1.03
#180		1.72
#181		1.91
#182		8.45
#183		0.22
#184		0.29
#185		1.38
#186		1.58
#187		2.49
#188		7.33
#189		2.86
#190	GGmCAToAtgcagataoATGTTmC	0.70
#191	GGmCAToAotgcagataoAoTGTTmC	4.29
CONTROL #4	GGGAGttacttgccaAmCTTG	0.24/0.36
#192	GGGAGoTtacttgccaAmCTTG	0.36
#193	GGGAGoToTacttgccaAmCTTG	0.28
#194	GGGAGoToToActtgccaAmCTTG	0.21
#195		0.23
#196		0.47
#197		0.41
#198	GoGoGAGttacttgccaAmCToToG	0.25
#199	GoGoGAGoTacttgccaAmCToToG	0.40
#200	GoGoGAGoToTacttgccAmCToToG	0.46
#201	GoGoGAGoToToActtgccaAmCToToG	0.47
#202		1.01
#203		2.02
#217		0.54
#218		2.22
CONTROL #5	TGmCmCTttaggattctAGAmCA	0.34/0.20
#219	TGmCmCToTtaggattctAGAmCA	1.08
#220	TGmCmCToToTaggattctAGAmCA	0.34
#221	TGmCmCToToToAggattctAGAmCA	0.81
#222	TGmCmCToToToAoGgattctAGAmCA	0.89
#223	TGmCmCToToToAoGoGattctAGAmCA	1.34
#224	TGmCmCToToToAoGoGoAttctAGAmCA	1.73
#225		7.61
#226		2.91
#238		1.78
#239		5.62
#240		8.39
CONTROL #6	mCmCAGGctggttatgamCTmCAG	0.88
#241		0.37
#242		0.30
#243		0.56
#244		0.71
#245		1.13
#246		1.47
#247		1.07
#248		1.09
CONTROL #7	TTATmCaattcaccaaGGAGmC	2.26
#249		0.93
#250		0.61
#251		1.12
#252		1.25
#253		1.68
#254		1.28
#255		2.18
#256		2.96
#257		6.34
CONTROL #8	ATGGAggtatgacatATAAT	0.94
#258	ATGGoAggtatgacatATAAT	1.56
#259	ATGoGoAggtatgacatATAAT	1.63
#260	AToGoGoAggtatgacatATAAT	0.97
#261	AoToGoGoAggtatgacatATAAT	0.99
#262	AoToGoGoAoGgtatgacatATAAT	0.27
#263	AoToGoGoAoGoGtatgacatATAAT	0.31
#264	AoToGoGoAoGoGoTatgacatATAAT	0.33
#265	AoToGoGoAoGoGoToAtgacatATAAT	0.53
#266	AoToGoGoAoGoGoToAoTgacatATAAT	0.88
#267		0.75
#268		1.44
#269		0.82
#270		0.48
#271		0.43
#272		1.37
#273		3.07
#274		0.92
#275		0.88
#276	GToAomCoTatcccatcacToGoAoAG	0.82
#277		2.03
#278		0.14
#279		1.04
#280		0.51
#281		1.19
#282		0.38
#283		0.14
#284		1.59
#285		1.55
#286		1.05
#287	mCmCTTmCoAoGoAoGoAoTtcaaTGmCTA	1.03
#288	GmCmCAAoToAoToToToGccccTmCmCmCmC	0.58
#289		1.09
#290		0.33
#291		0.49
#292	TGTAmCoAoToToToToGccctTAGmCT	0.62
#293	TTGmCToGoAoAoAoToTgtctmCAATT	1.53
#294		0.81
#295		1.46
#296	GmCmCTToAoAoAoGoToTacatTmCGTT	1.11
#297		0.47
#298		1.02
#299	ATGmCAoAoGoToToAoAacttATmCTG	0.42
#300		0.52
#301		0.48
#302		1.22
#303		0.53
CONTROL #1	GAGttacttgccaAmCT	1.32
#31	GAGoTotacttgccaAmCT	1.95
#32	GAGtoToacttgccaAmCT	1.62
#329	GAGoToToacttgccaAmCT	2.2
#330	GAGoTotacttgccoAoAmCT	12.02
#331	G*AGoTotacttgccaAmC*T	0.45
#332	G*AGtoToacttgccaAmC*T	1.08
#333	G*AGoToToacttgccaAmC*T	0.62
#334	G*AGoTotacttgccoAoAmC*T	1.25
CONTROL #2	GmCmCAGgctggttatgAmCTmCA	0.27
#81	GmCmCAGoGoctggttatgAmCTmCA	1.1
#118		0.91
#83		0.73
#110	GmCmCAGoGooGoctggttatoGoAmCTmCA	1.69
#335	G*mCmCAGoGoctggttatgAmCTmC*A	0.67
#336		0.66
#337		0.59
#338	G*mCmCAGoGoctggttatoGoAmCTmC*A	2.14
CONTROL #3	GGmCATatgcagataaTGTTmC	0.36
#339	GGmCAToAotgcagataaTGTTmC	0.51
#340	GGmCATaoTogcagataaTGTTmC	0.59
#341	GGmCAToAoTogcagataaTGTTmC	0.54
#191	GGmCAToAotgcagataoAoTGTTmC	3.37
#342	G*GmCAToAotgcagataaTGTT*mC	0.26
#343	G*GmCATaoTogcagataaTGTT*mC	0.37
#344	G*GmCAToAoTogcagataaTGTT*mC	0.47
#345	G*GmCAToAotgcagataoAoTGTT*mC	3.26

Example 4: Caspase 3/7 Activation HepG2 Cells

Caspase 3/7 Activation HepG2 cells were cultivated at app. 70% confluence in MEM medium with GlutaMax (Gibco #41090), supplemented with 10% heat inactivated fetal calf serum. Cells were detached with 0.25% Trypsin-EDTA solution (Gibco #25200056) and seeded into black, clear 96-well plates P37102-EP 78 (Corning #3904, NY, USA) at a density of 1×10⁴ cells/well. 24h post-seeding HepG2 cells were transiently transfected with Lipofectamine 2000 (Life Technologies #11668019) using 100 nM oligonucleotides dissolved in Opti-MEM (Gibco #31985). Caspase-3/7 activity was determined using the Caspase-Glo® 3/7 Assay (Promega Corporation, Madison WI, USA). Reconstituted Caspase-Glo® 3/7 reagent was added to the cells 24 hours post-transfection, incubated for 60 min, cell lysates were transferred into opaque 96-well plates (Corning #3600, NY, USA) before luminescence was determined on an Enspire multi-mode plate reader (Perkin Elmer) according to the manufacturer's instructions. The results are shown in Table 12, where the percentage (% assay window) indicates the degree of cell apoptosis based on vehicle (cells only treated with PBS). The higher the value, the higher apoptotic activity and thereby in vitro cytotoxicity. The negative control is a non-toxic ASO. Some values of tested ASOs are negative due to a lower background of these ASOs. Negative values are not cytotoxic while ASOs with values around 300 are slightly cytotoxic. ASO=antisense oligonucleotide.

TABLE 12
Caspase 3/7 assay results

		HepG2
		cells
		100
		nM
		ASO
		Caspase
		3/7
		act-
		ivation
		after
		24 h (%
CMP ID		assay)
NO	Sequence	window)	Target

CONTROL	GAGttacttgccaAmCT	294	MALAT1
#1
#29	GoAoGttacttgccaAmCT	0	MALAT1
#31	GAGoTotacttgccaAmCT	25	MALAT1
#32	GAGtoToacttgccaAmCT	25	MALAT1
#33	GAGttoAocttgccaAmCT	15	MALAT1
#34		219	MALAT1
#35	GAGttacoTotgccaAmCT	11	MALAT1
#36	GAGttactoTogccaAmCT	40	MALAT1
#37	GAGttacttoGoccaAmCT	128	MALAT1
#38		86	MALAT1
#39		71	MALAT1
#40	GAGttacttgccoAoAmCT	30	MALAT1
#41	GAGttacttgccaoAomCT	35	MALAT1
#42		109	MALAT1
#43	GAGttacttgccaAmCoT	68	MALAT1
CONTROL	mCTTtggcagaatgAGG	223	CERS2
#9
#309	mCTToTggcagaatgAGG	80	CERS2
#310	mCTToToGgcagaatgAGG	135	CERS2
#311	mCTToToGoGcagaatgAGG	20	CERS2
#312		32	CERS2
#313		16	CERS2
CONTROL	GmCTttggcagaatGAG	98	CERS2
#10
#314	GmCToTtggcagaatGAG	19	CERS2
#315	GmCToToTggcagaatGAG	16	CERS2
#316	GmCToToToGgcagaatGAG	51	CERS2
#317	GmCToToToGoGcagaatGAG	10	CERS2
#318		−8	CERS2
CONTROL	AmCatgctttcacagAATT	49	CERS5
#11
#319	AmCOAtgctttcacagAATT	2	CERS5
#320	AmCoAoTgctttcacagAATT	−6	CERS5
#321	AmCoAoToGctttcacagAATT	−10	CERS5
#322		−10	CERS5
#323		−11	CERS5
CONTROL	TTmCAttcacagacagGA	129	CERS6
#12
#324	TTmCAoTtcacagacagGA	39	CERS6
#325	TTmCAoToTcacagacagGA	75	CERS6
#326		20	CERS6
#327		31	CERS6
#328		18	CERS6

The data demonstrated that even a single TNA (PO) in 5′-flanks or DNA gap significantly reduced caspase induction across targets (MALAT1, CERS2/5/6).

Example 5: Thermal Melting (Tm) of Oligonucleotides Containing TNA (PO) Modifications Hybridized to RNA

The denaturation point (thermal melting=Tm) was measured according to the following procedure: Gapmer ASOs and complementary RNA were added to 20 mM disodium phosphate buffer, 200 mM NaCl and 0.2 mM EDTA (pH 7) resulting in a final concentration of 1.5 μM. Samples were heated to 95° C. for 5 min and then slowly cooled to room temperature over a period of 1 hour. Thermal melting curves were recorded at 260 nm on an Agilent Cary 3500 equipped with a Peltier Temperature Programmer using a temperature gradient that was increased by 5° C./min from 25° C. to 95° C. and then decreased to 25° C. The first derivatives of both curves were used to determine the melting temperature (Tm). The values were averaged over three heating and cooling curves.

TABLE 13
Thermal melting temperature (Tm) values

CMP		Tm
ID NO	Sequence	(° C.)

CON-	GAGttacttgccaAmCT	61.4
TROL #1
#10	GoAoGoToToActtgccaAmCT	59.5
#11		61.1
#12		61.3
#13	GoAoGoToToActtgccaamCT	57.3
#14	GoAoGoToToActtgccaacT	53.8
#15		58.5
#16		54.6
#17		58.2
#18		55
#22	GAoGttacttgccaoAomCT	54.3
#23	GAoGottacttgccaoAomCT	56
#24		52.8
#25		53.3
#26		53.8
#27		53.3
#28		54.3
CON-	GmCmCAGgctggttatgAmCTmCA	70.8
TROL #2
#80	GmCmCAGoGctggttatgAmCTmCA	68.9
#81	GmCmCAGoGoctggttatgAmCTmCA	69.3
#82		70.7
#83		71.2
#84		71.3
#85		71.6
#86		73.8
#87		73.7
#88		74.5
#89		74.8
#90		74.4
#105		67.6
#106		69.1
#107	GomCmCAGgctggttatgAmCTomCoA	69.3
#108		63.6
#109		63.5
#110	GmCmCAGoGoctggttatoGoAmCTmCA	72.0
#111		61.7
#112		60.8
#132	GomCmCAGgctggttatgAmCTmCA	70.6
#133		67.3
#134		66.5
#135	GmCmCoAGgctggttatgAmCTmCA	69.4
#136	GmCmCAoGgctggttatgAmCTmCA	69.1
#137	GmCmCAGoGctggttatgAmCTmCA	69.6
#138		70.1
#139	GmCmCAGgcoTggttatgAmCTmCA	70
#140	GmCmCAGgctoGgttatgAmCTmCA	71.6
#141	GmCmCAGgctgoGttatgAmCTmCA	70.5
#142	GmCmCAGgctggoTtatgAmCTmCA	70.2
#143	GmCmCAGgctggtoTatgAmCTmCA	71.3
#144	GmCmCAGgctggttoAtgAmCTmCA	70.5
#145	GmCmCAGgctggttaoTgAmCTmCA	72.4
#146	GmCmCAGgctggttatoGAmCTmCA	72.3
#147	GmCmCAGgctggttatgoAmCTmCA	71.1
#148		66.8
#149	GmCmCAGgctggttatgAmCoTmCA	67.9
#150		69.9
CON-	GGmCATatgcagataaTGTTmC	58.9
TROL #3
#151	GGmCAToAtgcagataaTGTTmC	55.6
#152	GGmCAToAoTgcagataaTGTTmC	58.3
#153	GGmCAToAoToGcagataaTGTTmC	59.3
#154		61.8
#163		61.8
#164		63.3
#165		61.5
#166		59.5
#167		65.0
#168		62.3
#183		53.7
#184		53.3
#185		48.9
#186		50.3
#187		50.4
#188		50.2
#189		50.9
#190	GGmCAToAtgcagataoATGTTmC	56.8
#191	GGmCAToAotgcagataoAoTGTTmC	57.9

The data demonstrated that TNA (PO) increased Tm when incorporated in the DNA gap. However, Tm was reduced when TNA was substituted for sugars with high binding affinity (MOE, LNA).

Example 6: Inhibition of MALAT1 Expression in Mice Administered Subcutaneously

To compare TNA to other sugars, a well-described MOE gapmer targeting MALAT1 was selected as a parent antisense oligonucleotide for the in vivo study. TNA substitution of MOE in flanks resulted in the following set of antisense oligonucleotides which were dosed subcutaneously with 10 mg/kg in mice.

TABLE 14
selected antisense oligonucleotides containing TNA for in vivo study in
mice

		IC50 [μM]	IC50 [μM]
CMP ID NO	Sequence	A549	LTK
CONTROL #2	GmCmCAGgctggttatgAmCTmCA	0.46	0.01
#346	GmComCoAoGgctggttatgAomCoTomCA	0.71	0.11
#347		2.02	ND
#100		0.35	0.05
#99		0.94	0.12
#76		0.38	ND
#78		0.52	ND
ND: not determined

In vivo experiments were conducted. The study was conducted with the approval of the local veterinary authority with strict adherence to the Swiss federal regulations on animal protection and to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Male C57BL/6 J mice (8 to 12 weeks old) were purchased from Charles River Laboratories, France. The mice were group housed in open cages and maintained on a 12:12-h light: dark cycle, with constant temperature (21-24° C.) and humidity (40-80%). Each cage was provided with unrestricted access to municipal water and sterilized food (Provimi Kliba 3436). All cages (except metabolism cages) were supplied with autoclaved sawdust bedding and environmental enrichments, which were applied to best practice animals' welfare standards and rotated weekly. The mice were acclimated for at least 1 week before the start of the study.

The animals were housed in groups of 3, water and a standard diet was supplied ad libitum. The vivarium was maintained at a constant temperature (23±1° C.) and humidity (40±5%) under a 12 h light: 12 h dark cycle (lights on at 08:00 h) throughout the study. Mice were dosed intravenously on days 0 and anesthetized (70% CO2/30% O₂) before termination by cervical dislocation on day 9. The treatment groups (n=4) received either 0.9% saline or saline-formulated gapmer administered by subcutaneous injection with a dosing of 10 mg/kg. At the end of the study, organs and plasma were collected at terminal time points. Plasma samples were stored at −20° C. for further processing and analysis. Organ samples were collected into homogenization tubes (Precellys® CK28, Bertin Instruments, France).

Total RNA from liver and kidney was isolated using the RNeasy kit (Qiagen) and quantification of RNA was done using TaqMan assays (Applied Biosystems). The reverse transcription reaction was carried out with random decamers, 0.5 mg total RNA and the M-MLV reverse transcriptase enzyme (Ambion) according to protocol for first strand complementary DNA (cDNA) synthesis. Depending on expression levels, cDNA was subsequently diluted 5 times in nuclease-free water before addition to the RT-PCR reaction mixture. The Applied Biosystems 7500/7900/ViiA real-time PCR instruments were used for amplification. Within each study, mRNA levels were normalized to actin, beta (Actb) or glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and presented as fold changes relative to average levels in saline controls.

As shown in FIG. 1, all antisense oligonucleotides resulted in a better reduction of MALAT1 RNA in liver compared to other tissues. In liver, at a dose level of 10 mg/kg the parent gapmer (CONTROL #2) demonstrated an RNA target reduction of approximately 70%. The corresponding PO-version (#346) performed substantially lower. The two TNA analogues with six POs introduced (#100 & #76) clearly outperform CONTROL #2. Even the two ASOs with eight phosphates inserted (#99 & #78) demonstrate equal in vivo efficacy compared to the all-PS parent. The all-PS TNA gapmer (#347) performed best with a target knockdown of ˜80%. TNA did not improve reduction of MALAT1 expression in lung and muscle as compared to MOE ASOs (CONTROL #2 & #346).

Tissue samples were collected and weighed during necropsy and stored at −80° C. in homogenization tubes CK28 (Precellys®) for subsequent bioanalytical examination by LC coupled to tandem mass spectrometry (LCMS/MS). Prior to extraction tissue homogenates were diluted 5-fold in mouse blank plasma. The quantification was performed against a mouse plasma calibration curve. Fifty microliters of calibration standards, quality control samples (freshly prepared in mouse plasma), and tissue homogenate samples diluted in mouse blank plasma were treated for protein denaturation with 150 μL of 4 M guanidine thiocyanate after addition of the internal standard (2000 ng/ml). A characterized 16-mer oligonucleotide (MW: 5460 Da), consisting of DNA nucleotides, LNA nucleotides, and a complete phosphorothioate backbone, was used as an internal standard in order to exclude possible variations during pipetting, solid-phase work-up, or LC-MS/MS sample injections. After vigorously mixing (20 min at 1,600 rpm), 200 μL of a H2O/HFIP/DIPEA solution (100:4:0.2, v/v/v) were added, followed by mixing (15 min at 1500 rpm). Then, a clean-up step was performed using solid-phase-extraction cartridges (Waters, OASIS HLB, 30 μm) after elution and evaporation to dryness (30-45 min at 40° C.) the samples were reconstituted in 200 μL of mobile phase (H2O/MeOH/HFIP/DIPEA [95/5/1/0.2, v/v/v/v]). After vortex mixing (10 min at 1500 rpm), an aliquot (20 μL) was injected into the analytical column (Waters, Acquity BEH C18, 1.7 μm, 50×2.1 mm kept at 60° C.). The analyte and internal standard were separated from matrix interferences using gradient elution from H2O/MeOH/HFIP/DIPEA (95/5/1/0.2, v/v/v/v) to H2O/MeOH/HFIP/DIPEA (10/90/1/0.2, v/v/v/v) within 4 min at a flow rate of 0.4 mL/min. Mass spectrometric detection was carried out on an AB-Sciex 6500+mass spectrometer using selected reaction monitoring (SRM) in the negative-ion mode. The selected ion reactions (m/z) were 680.6/94.8 and 658.9/94.8 for ASO 1 and ASO 5 respectively and 596. 1/94.8 for Internal Standard. Detection was accomplished utilizing ion spray MS/MS in negative ion SRM mode.

As shown in FIGS. 2A-H, antisense oligonucleotide contents in liver and kidney and lung and muscle at day 9 were quantified by using LCMS/MS and PK/PD relationships were demonstrated. TNA gapmer (#347) had the highest liver concentration which had a great correlation with its strong reduction of MALAT1 RNA in the liver. Antisense oligonucleotide #99 showed comparable efficacy albeit with lower liver content. Clearly, antisense oligonucleotides containing PO linkages were predominately eliminated into the kidney, probably due to lower affinity to plasma protein. As expected, all antisense oligonucleotides showed much lower concentrations in lung and muscle, consequently, which did not lead to a meaningful mRNA reduction in these two tissues.

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